Different types of PCR

Based on the thermal gradient pcr mechanism, the temperature gradient determined by number of wells is important factor for determination of amplification optimization. So the used maximum gradient temperature range between 15 oC and 30 oC advances the speed and specificity of PCR. The thermal gradient also provides optimization for the temperatures of denaturation, annealing or extension in one experiment. It is also useful for many applications containing the cloning and sequencing in molecular biology. [1] 

TOUCHDOWN PCR 

Touchdown pcr increases the specificity, sensitivity and yield of PCR cycles by using a special cycling program at which annealing temperature is decreased like 1-2 C for every second cycle. The initial annealing temperature is more than estimated Tm of the primers, while then it is decreased to calculated annealing temperature of the primers. This difference between the temperatures allows the exponential advantage of two-fold per cycle. It is useful in cDNA libraries and also the screening of single nucleotide polymorphism applications. [2] 

NESTED PCR 

Nested pcr is a method modified from standard PCR that avoids product contamination formed by amplification of undesired primer binding sites. In the nested pcr, primers are designed as two primer pairs including the one for outer fragment and one for the inner fragment such as outer forward – reverse primer and also inner forward – reverse primer. By this way, the large numbers of amplified outer fragments allow to continue the amplification of inner fragments resulting in the amplification with minimum contamination. [3] 

MULTIPLEX PCR 

Multiplex pcr technique depends on the combination of usage multiple primers and DNA polymerase enzyme for amplification of multiple targets DNA sequences in a single PCR thermal cycler. Multiple pcr performs the amplification of multiple targets by using single template or multiple templates. By this technique, the significant savings can be obtained in time with desired target DNA sequences. It is also used in many applications such as RNA detection, analysis of mutations and pathogen, etc. [4]  

 

HOT START PCR 

Hot start pcr technique allows decreasing non-specific amplification and also facilitates to set up the reaction at room temperature. In this technique, the ambient temperatures are used for preventing the polymerase activity which also activated at independent reaction temperatures resulting in the dissociation of it from its inhibitors and starts the polymerization. Hot start DNA polymerases are generally preferred for many applications needed a high degree of specificity. [5] 

REVERSE TRANSCRIPTION PCR 

If the starting material is RNA, the reverse transcription pcr is used by also helping of the reverse transcriptase enzyme. This enzyme transcribes the RNA from mRNA into cDNA used as template. This method is useful for many applications such as analysis of gene expression, identification of pathogen, etc. [6] 

REFERENCES 

  1. Gradient PCR Machine 

http://www.labcompare.com/General-Laboratory-Equipment/212-Gradient-PCR-Machine/ 

  1. Nature Protocols 3, 1452 – 1456 (2008)  

Published online: 21 August 2008 | doi:10.1038/nprot.2008.133 

Subject Category: Nucleic acid based molecular biology 

  1. Nested Primers for PCR 

Copyright 2002 Department of Biology, Davidson College, Davidson, NC 28036 

http://www.bio.davidson.edu/courses/genomics/method/nestedpcr.html 

  1. Multiplex PCR 

http://www.premierbiosoft.com/tech_notes/multiplex-pcr.html 

  1. D’Aquila, R.T. et al. Maximizing sensitivity and specificity of PCR by pre-amplification heating. Nucleic Acids Res. 19, 3749 (1991). 

Chou, Q., Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. Nucleic Acids Res. 20, 1717-1723 (1992). 

  1. Bustin S. (ed) (2004) A-Z of Quantitative PCR. IUL Biotechnology Series, International University Line, La Jolla, California. 

Expression Analysis of TAT mRNA

AIM   

In this experiment, aim was to analyze the mRNA expression in BL21 cells containing pGEX2T + TAT induced with IPTG by performing the RNA isolation at first followed by cDNA synthesis in which expressed mRNA was converted to cDNA in the presence of reverse transcriptase enzyme and lastly aim was to perform the Real Time PCR for controlling the expression level of TAT gene in the pGEX2T expression vector in the presence of SYBR Green dye. 

INTRODUCTION 

RNA (ribonucleic acid) is a nucleic acid which plays role in the transferring of genetic code or information from nucleus to ribosomes to make proteins, also to perform the transcription and translation processes. RNA has many similarities with DNA; actually, RNA has ribose sugar instead the deoxyribose sugar in DNA. It has uracil bases instead the thymine bases in DNA. Moreover, RNA is single-stranded molecule containing the shorter chains of nucleotides, while DNA is double-stranded molecule containing the long chain of nucleotides. RNA serves as the template for translation of genes to make proteins and also transcription into proteins. However; DNA plays role in the long-term storage of genetic information for the formation of new cell or organism by transmission of genetic information. DNA is capable of self-replication, while RNA can be synthesized from the DNA. Also, RNA is more resistant against the UV damage than DNA. There are three major types of RNA molecules acting in the gene expression including the messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA) [1] 

RNA isolation is the critical method in which the purification and extraction of RNA from the various biological samples including the bacteria, yeast, plant, cell culture, etc. RNA isolation is very sensitive because of the active RNases found in the cells which causes the RNA degradation. To prevent the degradation of RNA which will be purified, the isolation procedure must be performed in the presence of RNase-free solution [2]. During the RNA isolation procedure, also DNase treatment is applied to obtain only pure RNA molecules. The main logic of DNase treatment is its rapid degradation activity on the DNA molecules which removes the genomic DNA from the RNA samples by degradation of single and double stranded DNA molecule into single bases or oligonucleotides. Before the DNA treatment, DNA and RNA found as attached to the column together and by the rDNase treatment, digestion of bound DNA occurs and so only RNA molecules can be bounded to the silica membrane [3]. 

Followed by the RNA isolation, the purified RNA molecules as mRNAs can be converted into the cDNA which is the double-stranded DNA version of an mRNA molecule converted by reverse transcriptase enzyme. This enzyme converts the mRNA molecule into the single-stranded DNA by using the mRNA as template following by double-stranded DNA synthesis using the single-stranded DNA as template. In eukaryotes, processed RNA (mature mRNA) plays role as a template for the cDNA synthesis, while, in prokaryotes, transcribed mRNA (no processed RNA) directly plays role as a template for the cDNA synthesis [4]. mRNA cannot be generated directly and so it must be converted to cDNA for the biological studies. The cDNA version allows to easily expression of it into the expression vectors resulting in the high level of desired gene expression. Also cDNA version provides to analysis of expression amount of desired gene. It can be used for many applications including the Nothern analysis, RNase protection, DNA arrays, RT-PCR and in situ hybridization [5]. For the cDNA synthesis, mainly 3 primer types including the oligonucleotides, random primers and sequence specific primers can be preferred based on their functions, advantages and disadvantages. Oligonucleotides are available for the 1st strand cDNA synthesis via a reverse transcriptase. Their functions are the poly-A tail hybridization of mRNA. They have advantages about the generation of full length of cDNA from hybridized poly-A tail of mRNA and also better usage when the little beginning material is available. They have disadvantages about only amplification of gene with a poly-A tail. Random primers including the random hexadeoxynucleotides are available for the annealing along RNA transcript at multiple points. They have advantages about the annealing of all RNA types including tRNA, rRNA and mRNA and also yielding high level of cDNA. They have also disadvantages about the dilution effect of synthesized cDNA from all RNAs on the mRNA signals. Sequence specific primers are available for the specific mRNA sequences. They have advantages about formation of specific cDNA pool and high sensitivity, but also have disadvantages about the limited synthesis as one gene of interest [6] 

Real-Time PCR is a technique which used for the quantification of DNA or cDNA in a sample. It provides to detect the PCR amplification in the early stages of the reaction by measuring the reaction kinetics rather than in traditional PCR detection technique which detects the PCR amplification by agarose gel method at the end-point of PCR reaction. For this purposes, we can use the RT-PCR for the analysis of expression level of a target gene. RT-PCR has many advantages about the increasing the range of detection, even detection of two-fold changes and increasing of reporter fluorescent signals  resulting in the increasing the generated amplicons. RT-PCR detection is based on the usage of dyes such as SYBR Green or fluorescent reporter molecules such as Molecular Beacons, TaqManProbes, FRET Hybridization Probes and ScorpionR Primers. SYBR Green is a fluorogenic dye which binds to minor groove of double- stranded DNA molecule and so increases the intensity of fluorescent emissions. It is available for the rapid screening and also has lower costs than probe-containing assays. It also can be easily coupled with melting curve analysis to increase assay specificity. However; it has many disadvantages including the its less specificity due to detection of accumulated specific and non-specific PCR products, the generation of same fluorescence signals and non-availability for multiple gene. Moreover, TaqManProbes are fluorescent-labeled oligonucleotides which complementary to a region of the target gene. They have more specificity than SYBR Green dye due to accumulation of desired PCR product and also can be useful for the detection of multiple genes in the same well. But; they have a disadvantage about their higher costs. An amplification plot includes the information about the quantitative measurement of RNA or DNA samples in the presence of threshold line as the cycle threshold referred as Ct value at which the samples reach the more accurate exponential level. Baseline is also important term in the amplification plot which indicates the background noise level before the significantly amplification [7] 

Relative quantification is a method in which the analysis of changes in gene expression for a given sample related to other sample that can be an untreated or treated control sample such as treatment by IPTG or not [8]. In the quantification methods, two control groups are used including the negative and positive controls. Negative control is always performed without template and the genes in the operon are not expresses due to activity of a repressor protein and so the gene expression becomes switching off. Positive control is performed to compare between the samples in the presence of a housekeeping gene including the rRNA and actin which found in all cell types. In the case of positive control, the gene expression is switched on by the activity of an activator [9]. 

Melting curve also called the dissociation plot is performed with the heating of double-stranded DNA molecule with SYBR Green I dye at the end of the RT-PCR. So, the melting point (Tm) is reaching point at which detection of a sudden decreasing in fluorescence. In addition to this, this is plotted as temperature vs. change in fluorescence/ change in temperature [10]. 

MATERIALS&METHODS 

RNA Isolation 

RNA isolation procedure was performed by purified 2 total RNA samples for each group. 1.5 ml of overnight BL21 (DE3) cells were transferred into a 1.5 ml microcentrifuge tube and centrifuged at ≥12000 x g for 2 min followed by removal of supernatant. At first 100 µl of TE buffer was added into the tube containing cells by inverting of tube several times following by incubation of resuspended cells for 5 min at 15-25 oC. Then, 300 µl of Lysis buffer was added into the cells by vortexing for about 15 sec until the mixture was homogenized. 180 µl of ethanol (96-100%) was added into tube and mixed by pipetting. On the other hand, the GeneJET RNA Purification Column was prepared and the prepared lysate was transferred to the column and centrifuged at ≥12000 x g for 1 min followed by removal of supernatant. Then, 700 µl of Wash Buffer 1 was added to column and centrifuged at ≥12000 x g for 1 min followed by removal of flow-through. 600 µl of Wash Buffer 2 was added to column and centrifuged at ≥12000 x g for 1 min followed by removal of supernatant, again. After that, 250 µl of Wash Buffer 2 was added to column again and centrifuged at ≥12000 x g for 2 min followed by removal of supernatant and the empty tube was centrifuged again and after centrifugation, collection tube containing the flow-through was discarded and the GeneJET RNA Purification Column was transferred to a sterile 1.5 ml RNase-free microcentrifuge tube. Following this, 75 µl of water, nuclease-free was added to the center of the purification column membrane and centrifuged at ≥12000 x g for 1 min. Finally, the column was discarded and the certain amount of purified RNA was used for Nanodrop measurements and remained purified RNA was stored at -20 oC until use for other processes. 

cDNA Synthesis 

Based on the Nanodrop results of purified 2 RNA samples for each group, the required amounts of RNA were calculated according to 1 µg of RNA for the cDNA synthesis which shown below; 

For the 1st sample of our group;                   For the 2nd sample of our group 

1 µg = 1000 ng                                              1 µl         323.0 ng RNA 

1 µl         581.2 ng RNA                                  x           1000 ng 

X            1000 ng                                             x = 3.1 µl RNA 

X = 1.7 µl RNA 

 

To set up the reaction for cDNA synthesis, at first, 3.1 µl of RNA was added into the tubes containing 1 µl of random hexamers and also 7.9 µl of water was added into the tubes until the total volume has been reached to 12 µl. This prepared mixture was mixed gently and incubated at 65 oC for 5 min. On the other hand, other components were added into the mixture which shown below table; 

Components  Volume (µl) 
5X Reaction Buffer  4 
RiboLock RNase Inhibitor (20 U/ µl)  1 
10 mM dNTP Mix  2 
RevertAid M-MuLV RT (200 U/ µl)  1 
Total Volume  20 

 

By using the 44 µl of buffer and 22 µl of dNTP mix, a master mix was prepared and total 6 µl of this mixture was transferred into the PCR tubes of each group. Then, RiboLock RNase Inhibitor and RevertAid M-MuLV RT were added to tubes one by one and overall mixtures were incubated at 42 oC for 60 min. Finally, the reaction was terminated by heating at 70 oC for 5 min to perform the Real-Time PCR. 

Real – Time PCR 

To perform the Real-Time PCR processes, overall 80 µl of reaction mixtures were prepared as two mixtures including the TAT gene and also rRNA housekeeping gene (positive control) for each group. Firstly, 8 µl of both forward and reverse primers for own TAT was added into the tubes including 40 µl of SYBR Green mix. The same step was made for rRNA gene. Then, 24 µl of our own TAT gene was added into the mixture and also 24 µl of rRNA was added into the other tube. Followed by, 25 µl of both total mixtures for the TAT and rRNA gene were transferred into the 96-well plate one by one for each group and finally the Real-Time PCR was performed with following conditions; 

  • Initial denaturation: 95 oC for 10 min 
  • Denaturation: 95 oC for 15 sec 
  • Annealing: 48 oC for 30 sec                              40 cycles 
  • Extension: 72 oC for 30 sec 

Also conditions for melting curve; 

  • 55 oC for 2 min 
  • 95 oC continuous 

Thereby, the expression levels of mRNA for TAT gene were analyzed and recorded based on the Real-Time PCR graphs. 

RESULTS 

After performing the RT-PCR, we’ve obtained the many datas for the TAT gene and rRNA positive control housekeeping genes in terms of their Ct values and mean Ct values for cDNA 1 and cDNA 2 which found in tube 1 and tube 2, respectively. Also based on these values we’ve obtained the ratios which were shown below table 1 for cDNA 1 in tube 1 and table 2 for cDNA 2 in tube 2. 

Table 1: The total values obtained by performing the RT-PCR with the IPTG induction of cells for different times including 0h, 1h, 2h, 3h and 4h for TAT gene in the BL21 cells and rRNA positive controls in tube 1. In the table, the NTC means as the non-template which used for the control of PCR products and Ct values means indicating of expression levels of mRNA of TAT gene and positive contol rRNA gene. 

Type  Identifier  Ct Mean for TAT  Ct Mean for rRNA  TAT-rRNA  2^-(TAT-rRNA)  RATIO 
NTC  Ntc  37,31  32,59       
Unknown  0 hour  13,17  4,87  8,30  0,003173  1 
Unknown  1 hour  11,96  5,23  6,73  0,00942  2,969047 
Unknown  2 hour  13,13  5,61  7,52  0,005448  1,717131 
Unknown  3 hour  13,35  5,30  8,05  0,003773  1,189207 
Unknown  4 hour  16,14  3,92  12,22  0,00021  0,066064 

 

Based on the 2^-(TAT-rRNA) values in table 1, we’ve plotted a chart for the cDNA 1 by the different induction times as shown below figure;  

Figure 1: The plotted chart indicating the cDNA amount in tube 1 by the different induction times. 

 

Moreover, after performing the RT-PCR, we’ve obtained the amplification and dissociation plots for TAT gene and rRNA in tube 1 as shown below figures; 

 

Figure 2: The amplification plot for rRNA gene in tube 1 obtained by RT-PCR with different IPTG induction times including the 0h, 1h, 2h, 3h and 4h. Based on the figure, the green straight line indicates the threshold line obtained from the Ct values an also plateau and exponential phase were indicated in the figure. 

 

Figure 3: The dissociation plot (melting curve) for rRNA gene in tube 1 obtained by RT-PCR with different IPTG induction times including the 0h, 1h, 2h, 3h and 4h. Based on this figure, the melting temperature was 82.5 oC and also the melting point was indicated. 

 

 

 

 

Figure 4: The amplification plot for TAT gene in tube 1 obtained by RT-PCR with different IPTG induction times including the 0h, 1h, 2h, 3h and 4h. Based on the figure, the green straight line indicates the threshold line obtained from the Ct values an also plateau and exponential phase were indicated in the figure. 

 

Figure 5: The dissociation plot (melting curve) for TAT gene in tube 1 obtained by RT-PCR with different IPTG induction times including the 0h, 1h, 2h, 3h and 4h. Based on this figure, the melting temperature was between 81 and 82 oC and also the melting points were indicated. 

 

Table 2: The total values obtained by performing the RT-PCR with the IPTG induction of cells for different times including 0h, 1h, 2h, 3h and 4h for TAT gene in the BL21 cells and rRNA positive controls in tube 2. In the table, the NTC means as the non-template which used for the control of PCR products and Ct values means indicating of expression levels of mRNA of TAT gene and positive contol rRNA gene. 

Type  Identifier  Ct Mean for TAT  Ct Mean for rRNA  TAT-rRNA  2^-(TAT-rRNA)  RATIO 
NTC  Ntc  37,31  32,59       
Unknown  0 hour  15,68  5,72  9,96  0,001004  1 
Unknown  1 hour  13,02  3,47  9,55  0,001334  1,328686 
Unknown  2 hour  13,71  4,01  9,7  0,001202  1,197479 
Unknown  3 hour  12,32  4,03  8,29  0,003195  3,182146 
Unknown  4 hour  14,75  4,17  10,58  0,000653  0,650671 

 

Based on the 2^-(TAT-rRNA) values in table 2, we’ve plotted a chart for the cDNA 2 by the different induction times as shown below figure;  

Figure 6: The plotted chart indicating the cDNA amount in tube 2 by the different induction times. 

 

Moreover, after performing the RT-PCR, we’ve obtained the amplification and dissociation plots for TAT gene and rRNA in tube 2 as shown below figures; 

Figure 7: The amplification plot for rRNA gene in tube 2 obtained by RT-PCR with different IPTG induction times including the 0h, 1h, 2h, 3h and 4h. Based on the figure, the green straight line indicates the threshold line obtained from the Ct values an also plateau and exponential phase were indicated in the figure. 

 

Figure 8: The dissociation plot (melting curve) for rRNA gene in tube 2 obtained by RT-PCR with different IPTG induction times including the 0h, 1h, 2h, 3h and 4h. Based on this figure, the melting temperature was 84 oC and also the melting point was indicated. 

 

 

 

Figure 9: The amplification plot for TAT gene in tube 2 obtained by RT-PCR with different IPTG induction times including the 0h, 1h, 2h, 3h and 4h. Based on the figure, the green straight line indicates the threshold line obtained from the Ct values an also plateau and exponential phase were indicated in the figure. 

 

Figure 10: The dissociation plot (melting curve) for TAT gene in tube 2 obtained by RT-PCR with different IPTG induction times including the 0h, 1h, 2h, 3h and 4h. Based on this figure, the melting temperature was 82 oC and also the melting point was indicated. 

DISCUSSION 

In this experiment, we’ve used the BL21 (DE3) E.coli strain for the expression analysis, because, BL21 (DE3) cells uses the T7 RNA promoter which can be regulated by inducer as IPTG for the expression processes. In this experiment, we’ve aimed the analysis of TAT gene expression in BL21 cells containing pGEX2T + TAT induced with IPTG in different times such as 0h, 1h, 2h, 3h and 4h by comparing the TAT gene expression with rRNA gene expression which used as a positive control. At first, we’ve aimed to isolate RNA based on the kit procedure in the presence of many solutions TE buffer, Lysis buffer, Wash buffer, etc. In this case, we’ve used the TE buffer containing lysozyme enzyme for the breakdown of bacterial cell walls by hydrolysis of glycosidic bonds in the peptidoglycan backbone to improve protein or nucleic acid extraction efficiency. Following this, we’ve used the Lysis buffer containing B-mercaptoethanol for denaturation of RNases by reducing the disulfide bonds and destroying the native conformation required for the enzyme activity resulting in preventing of RNA degradation. Also, we’ve used the Wash buffer 1 and 2 containing ethanol for the removal of other cell components and many salts in the RNA isolation. At the end of the procedure, we’ve used the water with nuclease-free as elution buffer for the elution of bound RNA molecules into the solution. Followed by the RNA isolation, we’ve performed the cDNA synthesis in the presence of template RNA, random hexamers, water, buffer, dNTP mix, RNase inhibitor and a reverse transcriptase enzyme. After mixing of template RNA, random hexamers and water, we’ve incubated the mixture at 65 oC for 5 min to prevent the formation of secondary structure of RNA. Moreover, we’ve used the random hexamers for the cDNA synthesis in one direction by binding of primers to poly-A tail of mRNA molecule. We’ve used the RiboLock RNase Inhibitor to prevent the RNA denaturation in the mixtures and buffer to arrange the suitable conditions including pH and salt conditions for reverse transcriptase enzyme activity. Also we’ve used the reverse transcriptase enzyme as RevertAid M-MuLV RT for the synthesis of double-stranded DNA molecule from mRNA molecule. After cDNA synthesis we’ve aimed to perform the RT-PCR for controlling the expression level of TAT gene in the pGEX2T expression vector by comparing with expression of rRNA gene (positive control) in the presence of SYBR Green fluorogenic dye which binds the minor groove of double- stranded DNA molecule and so increases the intensity of fluorescent emissions by binding of DNA in amplification. Based on the RT-PCR results, we’ve obtained the total values for TAT gene and rRNA gene in tube 1 and 2 and we’ve observed the lowest Ct mean value for TAT gene after 4 hour of induction with IPTG unexpected results and highest value for after 1 hour of induction with IPTG unexpected result which shown in table 1. Because lower Cvalue means more template, so more gene expression for that sample and we could be expected the lower Cvalue after 4 hour of induction with IPTG and highest value at 0 hour without no induction with IPTG. Also we could be expected the lower Cvalue after 4 hour of induction with IPTG and highest value at 0 hour without no induction with IPTG for the rRNA gene as positive control and the results in table 1 for positive control were expected. Moreover, we’ve observed the lowest Ct mean value for TAT gene after 3 hour of induction with IPTG unexpected results and highest value for 0 hour of induction with IPTG expected result which shown in table 2. Because lower Cvalue means more template, so more gene expression for that sample and we could be expected the lowest value after 4 hour of induction with IPTG and highest value at 0 hour without no induction with IPTG. Also we could be expected the lower Cvalue after 4 hour of induction with IPTG and highest value at 0 hour without no induction with IPTG for the rRNA gene as positive control and the results in table 2 for positive control. But, we’ve observed the lowest value after 1 hour induction with IPTG, unexpected result. Based on these values the obtained amplification plots for TAT gene and rRNA gene in tube 1 and 2, the results were expected.  The green straight lines in the amplification plots indicated the threshold lines. Moreover, we’ve plotted the dissociation plots (melting curves) to control the amplification of TAT gene and based on the dissociation plot for rRNA gene in tube 1, we’ve observed one pick, expected result and determined the melting temperature as 82.5 oC which shown in figure 3. Based on the dissociation plot for TAT gene in tube 1, we’ve observed two picks, unexpected result and determined the melting temperature as between 81 and 82 oC a which shown in figure 5.  Based on the dissociation plot for rRNA gene in tube, we’ve observed two picks, unexpected result and determined the melting temperature as 84 oC which shown in figure 8. Based on the dissociation plot for TAT gene in tube 2, we’ve observed two picks, unexpected result and determined the melting temperature as 82 oC a which shown in figure 10.  Actually, we could be expected to observe one pick in the each melting curve, especially in melting curve for rRNA gene. Because, if we observe one pick, we can be sure about the amplification of TAT gene or rRNA gene, but if we observe two picks, we cannot be sure about the amplification of TAT gen or rRNA gene. The reason of formation of two picks can be contaminations due to SYBR Green dye. Lastly, we’ve plotted the charts indicating cDNA amount in tube 1 and tube 2 by using the 2^-(TAT-rRNA) values in table 1 and 2. Based on the chart for tube 1, we’ve observed the highest cDNA amount after 1 hour induction with IPTG and lowest after 4 hour induction with IPTG. These results were unexpected. We could be observed the highest cDNA amount after 4 hour induction with IPTG and lowest amount at 0 hour without any induction with IPTG. Also, based on the chart for tube 2, we’ve observed the highest cDNA amount after 3 hour induction with IPTG and lowest after 4 hour induction with IPTG. These results were unexpected. We could be observed the highest cDNA amount after 4 hour induction with IPTG and lowest amount at 0 hour without any induction with IPTG. 

REFERENCES 

  1. DNA vs. RNA 

http://www.diffen.com/difference/DNA_vs_RNA 

  1. Wilfinger WW, Mackey K, and Chomczynski P (1997) Effect of pH and ionic strength on the spectrophotometric assessment of nucleic acid purity. Biotechniques 22:474­481. 
  1. Vanecko, S, Laskowski, M (1961). J Biol Chem. 236, 3312-16. 
  1. Reverse Transcription (cDNA Synthesis) 

https://www.neb.com/applications/cloning-and-synthetic-biology/dna-preparation/reverse-transcription-cdna-synthesis 

  1. Why Isolate cDNAs? 

http://www-users.med.cornell.edu/~jawagne/cDNA_cloning.html#Why Isolate cDNAs 

  1. RNA Priming Strategies 

https://www.thermofisher.com/tr/en/home/life-science/pcr/reverse-transcription/rna-priming-strategies.html 

  1. Real-Time PCR Vs. Traditional PCR 

http://www6.appliedbiosystems.com/support/tutorials/pdf/rtpcr_vs_tradpcr.pdf 

  1. Absolute vs. Relative Quantification for Qpcr 

https://www.thermofisher.com/tr/en/home/life-science/pcr/real-time-pcr/qpcr-education/absolute-vs-relative-quantification-for-qpcr.html 

  1. 82 Miller, J. and Reznikoff, W. (1978).  The Operon.  Cold Spring Harbor Symp. Quant. Biol.. 
  1. Dwight Z, Palais R, Wittwer CT. (2011) uMELT: prediction of high-resolution melting curves and dynamic melting profiles of PCR products in a rich web application. Bioinformatics. 27(7):1019–1020. 

 

BACTERIAL GROWTH MEASUREMENTS: VIABLE COUNT

INTRODUCTION 

The aim of this laboratory hour was to provide both theoretical knowledge and practical skills about the process of counting different organisms present in a specific sample.  

Process of counting can be realized by using either direct or indirect method. There are some factors that affect the counting, such as: pipetting, optimized incubation time, maintenance of homogeneity and a proper amount of the mixture [1]. 

Direct method includes two different techniques: total cell count and viable count, where the only difference between them is that in the viable count, only the cells that have the potential to give rise to new colonies are counted, while the total cell count technique involves counting both living cells and dead cells. Viable count is known also as plate count or colony count method Viable count technique is unable to give every time accurately the total number of the viable cells, because of the possibility it exists that the groups of cells get disrupted before being plated or the cells to get clumped and form a colony together. In order to take in account the possibility that the colony forming unit can contain one or more cells, the number of the viable cells is calculated as colony forming unit (cfu). Viable count can be realized either by spread plate method or pour plate method [2]. 

Serial dilution of bacterial culture is a crucial process in the process of enumeration a sample with unknown number of bacteria and it is realized by diluting the sample either with broth, saline solution or phosphate buffer in the ratio 1:9. The range of the number of colonies is 30-300 and it is an optimized value, as a number less than 30 colonies does not fulfill the statistical requirements, while a number bigger than 300 would cause colonies to compete for nutrition and could also lead to the suppress of the colony growth [3]. 

 

MATERIALS & METHOD 

Dilution of Bacteria Cultures for Spread Plate and Pour Plate Techniques 

Materials 

  • Bunsen burner 
  • Pipettes & tips 
  • E. coli culture 
  • Test tubes consisting of LB broth 
  • Vortex 

Method 

1 ml from the Eppendorf that holds overnight cultured E. coli is taken by using the micropipette.  Tube where 9 ml of broth is positioned is opened and sterilized through the flame of the Bunsen burner. Then 1 ml of E. coli is poured inside the broth and the tube`s mouth is sterilized again through the flame. Tube is vortexed and labeled with the specific data about the experiment. The procedure of dilution is repeated 7 times by using each time 1 ml from the tube that was diluted and vortexed in the preceding step.  

 

Pour Plate Technique 

Materials: 

  • Water bath  
  • Diluted E. coli culture 
  • Pipettes and tips 
  • 15 ml LB agar in tubes 
  • Petri dishes 

Method  

Initially, 1 ml of the diluted sample of E. coli (10-4, 10-5, 10-6, 10-7) is positioned in the Petri dish.  Afterwards, 15 ml melted agar that was molten in the water bath at a temperature of 50OC, is added in the Petri dish. The plate is covered and moved over the bench in order that the samples inside get swirled. After that, the Petri dish is positioned there till the solidification of agar occurs and then placed in the incubator for 24 hours at a temperature of 37OC. 

Spread Plate Technique  

Materials 

  • Drigalski spatula 
  • Bunsen burner 
  • Pipettes and tips 
  • LB agar plates 
  • Diluted E. coli cultures 

Method 

From the diluted samples (10-4, 10-5, 10-6, 10-7) 0.1 ml is taken by using the micropipette and it is thrown into the agar plate. The Drigalski spatula is first sterilized inside the alcohol and later through the fire. After it cools down, this spatula is used to spread the inoculums throughout the area of Petri dish. The plate is covered and placed over the bench for a short period of time so that the inoculums get absorbed by the agar. Afterwards, the Petri plate is positioned upside down and incubated for 24 hours under the temperature of 370C. 

 

Standard Plate Count of Milk 

Materials 

  • Milk 
  • LB agar plates 
  • Pipette and pipette tips 
  • Drigalski spatula 
  • Tubes consisting of 9 ml saline solution  

Method  

1 ml from the milk sample is taken by micropipette and poured inside the tube that contains 9 ml of saline solution whose mouth was sterilized through the Bunsen burner. The sample is vortexed for a short period of time. This process is repeated by transferring 1 ml of sample of the preceding step`s culture to the next buffer till the dilution of 10-4. After the dilution procedure is over, the dilutions 10-1, 10-2, 10-3, 10-4 plates are spread by using Drigalski spatula. The plates are covered and positioned over the bench for a short period of time till the inoculums are absorbed by agar. Afterwards, the plates are placed in incubator for 24 hours under the temperature of 37oC. 

 

RESULT 

Plate count about each dilution factor  Cigdem` s group 

(raw milk) 

Ayhan` s group 

(raw milk) 

Nergiz` s group 

(pasteurized milk) 

Oyku` s group 

(raw milk) 

Deniz` s group 

(pasteurized milk) 

Elvan`  s  

group 

(raw milk) 

10-2    314  0  2    43 
10-3    32  0  3  0  1 
10-4  11  2  0  4  0  0 
10-5  0  1  0  2  0  0 
10-6  0        0   
10-7  0        0   

Table 1: Serial dilutions of the samples of raw milk and pasteurized milk and series of plates inoculated with diluted cultures 

Dilution factor  10-2  10-3  10-4  10-5  10-6  10-7 
Plate count  314; 0; 43  32; 0; 0; 1  11; 2; 0; 2; 0; 0  0; 1; 0; 4; 0; 0  0; 3; 0  0; 2; 0 
Colony-forming units  (314+43)/2 x1/10-2= 

1.79 x 104 

32 x 1/10-3= 

3.2 x 104 

0 (as no one of the values are in the range [30;300]  0 (as no one of the values are in the range [30;300]  0 (as no one of the values are in the range [30;300]  0 (as no one of the values are in the range [30;300] 

Table 2: CFU calculations of the standard plate count of the milk by using the formula [CFU= Plate Count x Dilution Factor]. The values are represented either as duplicate or triplicate, because of the experiment performed by the different lab groups. 

Dilution factor  10-4  10-5  10-6  10-7 
Plate count  >300  >300  135  13 
Colony-forming units  >300 x 1/104= 

More than 3 x 106 colony forming units 

>300 x 1/10-5= 

More than 3 x 107 colony forming units 

135 x 1/10-6=1.35 x 108  As it is not in the range [30;300] it is not calculated 

Table 3:  CFU`s calculations of the serial dilutions of samples inoculated by the spread plate technique 

Figure1: The representative image of bacteria inoculated by the spread plate technique with diluted factors 10-4, 10-5, 10-6, 10-7. It is seen that as the dilution factor decreases, number of bacteria decreases too. 

 

 

Figure 2: The representative images of the growth of bacteria in the standard plate count of the raw milk of Ayhan` s group ordered according to the dilution factor, specifically: 10-2; 10-3; 10-4; 10-5. It is seen that there is a decrease in the growth of bacteria proceeding from the left to right samples.  

 

 

Figure 3: The representative image of the growth of bacteria inoculated by the pour plate technique. The used diluted samples are 10-4, 10-5, 10-6, 10-7. The growth of bacteria increases proceeding from the left to the right samples. 

 

DISCUSSION  

By comparing the data from the standard plate count of the raw milk and pasteurized milk, it is seen that there is a relationship between the number of bacteria and whether the milk is raw or pasteurized. In the pasteurized milk, the plate count leaded to a number of 0 bacteria. Additionally, in the raw milk it is seen that as dilution factor`s value decreases, the number of bacteria decreases. In the calculations of the colony forming units, most of the calculations are not performed because the values of bacteria are outside the range [30; 300].  

In the pour plate method, it is observed that most of the bacteria have accumulated in the upper part of the medium and this occurs, because they have a high requirement of oxygen. The other part has accumulated in the down part of the medium, because they are micro-aerophiles and do not need lots of oxygen.  

In the spread plate method, it is seen that as the dilution factor decreases, number of bacteria decreases too. The reason for that is that as the dilution process is made the ratio of the sample to the medium continues to proceed in this form: 1/100, 1/1000, 1/10000.  

 

REFERENCES 

  1. http://biolabs.tmcc.edu/Micro%20Web/BacterialCounts.pdf retrieved 31.03.2016 from 
  1. https://www.boundless.com/microbiology/textbooks/boundless-microbiology-textbook/culturing-microorganisms-6/counting-bacteria-63/viable-cell-counting-384-5695/ retrieved 31.03.2016 from  
  1. http://textbookofbacteriology.net/growth_2.html retrieved 31.03.2016 from 

 

 

TA Cloning of TAT

AIM 

Aim of this experiment was to amplify TAT gene by PCR technique and to purify the obtained TAT gene fragments by applying the gel extraction method such as Thermo Scientific GeneJET PCR Purification Kit for the TA-cloning of TAT.  

INTRODUCTION 

Polymerase Chain Reaction 

PCR (polymerase chain reaction) is a common laboratory technique which provides the amplification of a specific segment of DNA sequence from a mixture of DNA molecules. The polymerase chain reaction is performed into three stages including the denaturation, annealing and elongation or extension which are also called PCR cycle. In the denaturation stage, applying the heat (more than 90 oC) separates the double-stranded DNA into two single-stranded DNA by breaking down the weak hydrogen bonds between the bases. However; the stronger bonds between deoxyribose and phosphates do not break. In the annealing stage, the DNA mixture is cooled (40 oC and 65 oC) allowing the primer binding and annealing to target complementary sequence in the template DNA. In the elongation stage, the temperature is increased to 72 oC which is optimal temperature for DNA polymerase activity allowing the DNA polymerase to synthesize new strand of DNA by also adding the nucleotides (dNTPs) onto the end of the extending primers. Synthesis of new strand always starts at 3’ end of the primer and continues in the 5’ to 3’ direction. As a result, two new DNA strands identical to original target can be amplified. The PCR cycle is repeated many times (30-40) and by this way, the synthesized products in a given cycle can serve as templates for the next cycles and so the number of target DNA copies become about double in every cycle [1]. 

The PCR reaction is set up with essential components including the a template DNA, a suitable DNA polymerase enzyme like Taq DNA polymerase, a buffer solution for the enzyme, magnesium chloride, dNTP mix, forward and reverse primers and water. By combining of all these components, a reaction mixture is prepared for performing the PCR. In this case, a template DNA also known as obtained DNA from samples includes target sequence of interest which will be amplified.  The DNA polymerase is an enzyme which synthesizes a new strand of DNA also via sequential addition of nucleotides [2]. Taq DNA polymerase is a highly processive thermostable enzyme which synthesized a new strand of DNA by addition of dNTPs onto the end of an annealed primer. For its activity, it needs to magnesium ions in which magnesium chloride. Taq DNA polymerase also adds the single deoxyadenosine (A) to 3’ end of the PCR product which is ligated with 3’ T overhangs of the cloning vector for the TA cloning process [3]. Buffer is a salt-solution which provides the optimal pH and salt condition for DNA polymerase and other reaction components. Primers including the forward and reverse primers are small segments of single stranded DNA (15-30 bases long) which provide annealing to their complementary sequence on the template strand. They provide initiation sites for DNA synthesis by binding to end of the target DNA sequence. In the PCR technique, magnesium as a cofactor is used in the magnesium chloride form and it provides the optimum activity for DNA polymerase enzyme. Lastly, dNTPs are four nucleotides which extend an annealed primer by acting of DNA polymerase [2]. 

For the PCR optimization, each required component must be mixed with appropriate concentrations of components during the PCR reaction. Buffer concentration including the pH and salt concentration must be in an appropriate concentration in which high salt conditions damage the DNA by denaturation resulting in the failure of the reaction and also low pH conditions affect the enzymatic activity (not worked). Also primer concentration must be appropriate at which high primer concentration causes mispriming and accumulation of nonspecific products resulting in decreasing the specificity and at which low concentration causes the completing reaction early resulting in the less yield of desired product. Moreover, high enzyme concentration causes the amplification of non-specific annealed primers resulting in the decreasing the specificity and low enzyme concentration causes low amplification of annealed primers resulting in the less yield of desired product. High DNA concentration causes the false priming resulting in the less DNA synthesis and low DNA concentration causes the less yield of desired product. The last important factor in PCR optimization is water concentration which arranges the optimal pH and salt concentration by dilution of buffer like from 10X to 1X [4].    

Gel Extraction 

Gel extraction is a method allowing the high purity of DNA which is used to isolate and purify the desired fragment of DNA from other bigger and smaller DNA (non-specific) based on their size by cut out of the visualized band on the agarose gel with performing the specific gel extraction processes especially spin-column kit procedure [5][6]. 

 

 

MATERIALS&METHODS 

PCR Reaction for TAT Gene 

For the PCR reaction, the isolated pCV-1 plasmid including the TAT gene from previous experiment was used as a template. The reaction mixture was prepared as 4 reactions for preventing losing of materials in the presence of any problems. Thereby, required amounts of all PCR components waited on ice were mixed (except the DNA) for the four reactions master mixture in order as shown below table:  

Table 1: The required amounts of PCR components for one reaction and four reactions to set up PCR reaction mixtures 

Component  Volume (µl)  Master Mix Volume (4 rxn) 
Nuclease-free dH2O  13.5 µl  54 µl 
10X Taq Buffer  2.5 µl  10 µl 
dNTP mix, 10 mM each  1 µl  4 µl 
25 mM MgCl2  2.5 µl  10 µl 
Forward primer (2 pmol)  2 µl  8 µl 
Reverse Primer (2 pmol)  2 µl  8 µl 
Taq Polymerase (5u/µl)  0.5 µl  2 µl 
DNA ng/µl  1 µl  Total 24 µl for each tube 

+ 1 µl of DNA 

TOTAL  25 µl 

 

After set up of master mix, 24 µl of mixture without the DNA sample was transferred into 2 PCR tubes for each group and then 1 µl of DNA was added into the each tube as the total volume of reaction mixture has been reached to 25 µl. The PCR tubes holded on ice was gently flicked to mix the solution and briefly centrifuged. Finally, the PCR tubes were placed into PCR machine and PCR cycles were performed under certain conditions as below: 

 

  • Initial Denaturation: 94 oC for 3 min 
  • Denaturation: 94 oC for 30 sec 
  • Annealing: 53 oC for 30 sec            30 cycle 
  • Extension: 72 oC for 45 sec 
  • Final Extension: 72 oC for 5 min 

Agarose Gel Electrophoresis 

After performing the PCR reaction, the PCR products including the amplified TAT gene and other molecules were loaded into an agarose gel. Before loading the samples, 0.8% agarose gel was prepared previously. In this case, to prepare 8% agarose gel, 1.2 g agarose was weighed and it was solved into 150 ml TAE Buffer by applying the heat and then cooled for addition of intercalating agent ethidium bromide. After preparation of agarose gel, all of 25 µl of PCR products were loaded into three gel wells with mixing 5 µl of 6X loading dye as the last concentration of loading dye comes to 1X which calculation for it is shown as below: 

Stock concentration of loading dye = 6X 

Final concentration of loading dye = 1X     So; 

M× V1 = M× V2 

1X × (25 µl +?) = ? × 6X                    

? = 5 µl of loading dye required 

The gel was run by 100 V electric current for 30 min. Finally, the gel slice including the desired DNA fragments were removed from the agarose gel by cut out of them under the UV light and the removed gel slices (2 gel slices for each group) were weighed for the next processes as gel extraction. 

Gel Extraction  

Firstly, the weight of cut out gel slices containing the fragment were determined as 0.2157 g ~ 216 mg for our group and 0.1666 g ~ 167 mg for other subgroup. The gel extraction processes were performed by Thermo Scientific GeneJET PCR Purification Kit. Then 1:1 volume of Binding Buffer, 216 µl of Binding Buffer was added into the gel slice and the gel mixture was incubated at 60 oC for 10 min until the gel slice was completely dissolved. In this stage, the tube with gel slice was mixed by inversion at certain intervals to facilitate the melting of gel slice. On the other hand, during dissolving, the color of solution was checked to control pH level for DNA binding. The color of our solution was yellow which indicates an optimal pH for DNA binding. But, if the color of solution was orange or violet, 10 µl of 3 M sodium acetate, pH 5.2 solution will be added into the gel mixture for obtaining the yellow color. After the gel slice completely dissolved, a 1:2 volume of 100% isopropanol, 216 µl of 100% isopropanol was added into solubilized gel solution and it was mixed. Then, up to 800 µl of the solubilized gel solution was transferred to the GeneJET purification column and centrifuged for 1 min. After the centrifugation, the supernatant was removed and the column was placed back into the same collection tube. After that, 700 µl of Wash Buffer added into the GeneJET purification column and centrifuged for 1 min and again the supernatant was removed. The empty GeneJET purification column was centrifuged for an additional 1 min to completely removal of residual wash buffer. Then, the GeneJET purification column of one subgroup was transferred into a clean 1.5 ml microcentrifuge tube and 45 µl of Elution Buffer was added to the center of the purification column membrane and centrifuged for 1 min. After centrifugation, the same Elution Buffer (45 µl) was used for our group’s GeneJET purification column and again centrifuged for 1 min. Finally, the purified DNA of each subgroup of our group was collected into the same tube by discarding the GeneJET purification columns and some of purified DNA was stored at -20 oC and some (2 µl) was used for Nanodrop measurements. 

Nanodrop Measurements 

For the Nanodrop measurements, firstly, 2 µl of dH2O was put into the pit of Nanodrop machine for cleaning the pit and it was dried. Then 2 µl of solvent of DNA (TE/ dH2O) was put into the pit as a blank in order to calibrate and pit was dried again. 2 µl of sample (DNA in TE/dH2O) was put into the pit and absorbance values including the A260/A280 and A260/A230 and also total DNA amount (ng/ µl) were determined and the results were recorded. 

 

RESULTS 

By performing the polymerase chain reaction we’ve amplified the TAT gene and also we’ve visualized the TAT gene fragments for each group by applying the agarose gel electrophoresis method and so, we’ve examined the PCR products on the agarose gel under the UV light which shown in below figure; 

   

Figure 1: The agarose gel image of amplified TAT genes results of the performing the PCR reaction in the presence of a ladder with 1 kb length bond size. TAT gene has a 263 bp length and so, the bands at approximately 300 bp indicate the amplified TAT genes. In addition to this, G1-1, G1-2 and G1-3 DNA bands indicate that the 1st group’s amplified PCR products including TAT gene with approximately 300 bp. G2-1, G2-2 and G2-3 DNA bands indicate that the 2nd group’s amplified PCR products including TAT gene with approximately 300 bp. G3-1, G3-2 and G3-3 DNA bands indicate that the 3rd group’s amplified PCR products including TAT gene with approximately 300 bp which not work the PCR correctly. Because, the bands are not very clear on the gel image especially the indicated band of G3-3 as shown above figure. G4-1, G4-2 and G4-3 DNA bands indicate that the 4th group’s amplified PCR products including TAT gene with approximately 300 bp. G5-1, G5-2 and G5-3 DNA bands indicate that the 5th group’s amplified PCR products which not worked PCR and so we’ve not observed any bands for TAT gene with approximately 300 bp in the 5th group’s gel results. 

After the agarose gel electrophoresis each group has cut out the own gel slice from the agarose gel under the UV light and performed the gel extraction method to isolate and purify the desired fragment of DNA from other bigger and smaller DNA molecules (non-specific). Thereby, at the end of the gel extraction processes, we’ve measured the absorbance values including the A260/A280 and A260/A230 ratios for checking the purities of extracted DNA molecules and also total DNA amount (ng/ µl) by using the Nanodrop Spectrophotometry and recorded the datas which shown below table: 

 

Table 2: The measured results of purified DNA molecules including the TAT gene with absorbance ratios and also total amount after by performing the gel extraction processes  

  Gel Extraction Method 
A260/A280  1.88 
A260/A230  0.05 
Total DNA (ng/µl)  20.7 ng/µl 

 

Based on the Nanodrop results, we’ve checked the purities of collected DNA molecules for our group and observed that the extracted DNA molecules were pure when we’ve examined the A260/A280 ratio, whereas when we’ve look at the A260/A230 ratio, we’ve observed that the alcohol contamination. Also, we’ve determined the total DNA amount as 20.7 ng/ µl which was unexpected result. We’ve used this data for the calculation of required DNA amount which will be used for the ligation reaction. 

DISCUSSION 

In this lab session, we’ve aimed the amplification of TAT gene by performing the polymerase chain reaction in the presence of required reaction components and we’ve aimed the controlling of amplified TAT genes by agarose gel electrophoresis method followed by the cut out of the gel slice with desired DNA fragment, we’ve aimed to purify the DNA molecules with amplified TAT gene fragments by applying the gel extraction method such Thermo Scientific GeneJET PCR Purification. For this purposes, at first we’ve performed the polymerase chain reaction by preparation of master mix including four PCR reaction mixtures for preventing losing of materials in the presence of any problem such as in pipetting, mixing, etc. In the PCR reaction, we’ve used the isolated pCV-1 plasmid from previous experiment as a template which includes target sequence of interest as TAT gene. Moreover, we’ve used the essential amounts of other PCR components such as MgCl2, dNTP mix, forward and reverse primers, Taq DNA polymerase enzyme and buffer for Taq DNA polymerase as shown in table 1. We’ve used the Taq DNA polymerase for synthesizing the new strand of DNA with also sequential addition of nucleotides like dNTPs. Also we’ve used it for production of PCR product with 3’ end which is necessary for TA cloning processes. The Taq DNA polymerase activity depends on the magnesium ions and buffer conditions. Magnesium ions interact with negatively charged molecules due to its positive charge in the reaction such as template DNA, primers and also dNTPs [2]. Moreover, we’ve used the buffer solution for optimal pH and salt condition for DNA polymerase activity and stabilization of other components. Also we’ve used the forward and reverse primers extended by dNTPs for annealing to their complementary sequences on the template strand. When we’ve set up the PCR reaction, we’ve mixed the PCR components in an order as water, buffer, dNTP mix, magnesium chloride, forward and reverse primers, Taq DNA polymerase and lastly template DNA. We’ve mixed the water and buffer at first because the water decreases the harmful effect of buffer solution on enzyme activity and other components by diluting the buffer. We’ve added the enzyme and DNA lastly for preventing the any damage to enzyme or starting reaction from nonspecific regions at which primers bind randomly. At the end of this stage, we’ve total 25 µl of reaction mixture and performed the PCR cycles under the suitable PCR conditions. After performing the PCR reaction, we’ve prepared the 0.8% agarose gel which 1.2 g of agarose was solved in 150 ml TAE buffer by applying the heat and then the solution cooled before addition of ethidium bromide intercalating agent for preventing the its harmful effect. We’ve prepared the 0.8% agarose gel for the separation of large DNA fragments. So, we’ve loaded the all of PCR products into three gel wells with mixing 6X loading dye as the last concentration of loading dye as 1X. We’ve used the loading dye including the glycerol and bromophenol blue to sinking of the samples into the well by glycerol and visualization of DNA migration by bromophenol blue. Also we’ve used the ethidium bromide intercalating agent for visualization of DNA bands under the UV light. After the agarose gel electrophoresis, we’ve cut out the gel slices (two gel slices for each group) for the gel extraction processes. Based on the agarose gel image shown in figure 1, we’ve expected to observe bands at 263 bp (~ 300 bp) length for amplified TAT genes. In addition to this, we’ve observed the approximately 300 bp bands for the G1-1, G1-2 and G1-3 DNA bands which indicate the 1st group’s amplified PCR products and this result was expected. We’ve observed the approximately 300 bp bands for the G2-1, G2-2 and G2-3 DNA bands which indicate the 2nd group’s amplified PCR products. But the band in G2-1 was clearer than G2-2 and G2-3. The reason of these less clear bands can be excess amount of enzyme and long extension time [8]. We’ve observed the approximately 300 bp bands for the G3-1, G3-2 and G3-3 DNA bands which indicate the 3rd group’s amplified PCR products. But the band in G3-2 was clearer than G3-1 and G3-3. The reason of these less clear bands can be excess amount of enzyme and long extension time [8]. We’ve observed the approximately 300 bp bands for the G4-1, G4-2 and G4-3 DNA bands which indicate the 4th group’s amplified PCR products and this result was expected. Moreover, we’ve not observed any bands for G5-1, G5-2 and G5-3 DNA bands which indicate that the 5th group’s amplified PCR products. This means that the PCR cycles have not performed correctly or the agarose gel electrophoresis has not performed correctly even TAT gene fragments have amplified. This result was unexpected. The reason of this can be too short extension time, very high annealing temperature, and inappropriate buffer causing the enzyme or DNA denaturation, low primer concentration, long denaturation time and inappropriate amount of magnesium ions [7]. After the agarose gel electrophoresis, we’ve performed the gel extraction method by using the Thermo Scientific GeneJET PCR Purification Kit. We’ve performed the gel extraction method because, the PCR mixture was not used directly in cloning and in addition to this and to remove the other bigger and smaller nonspecific DNA molecules and we’ve isolated and purified the desired fragment of DNA. For this purposes, at first we’ve determined the weights of the gel slices containing the fragment as 0.2157 g ~ 216 mg for our group and 0.1666 g ~ 167 mg for other subgroup. Based on these weights we’ve performed other processes. We’ve used Binding Buffer for dissolving the gel slice by incubation at 60 oC. In this case, the concentration of gel slice was depending on the buffer, heat and incubation time. On the other hand, during dissolving, we’ve checked the color of solution to control pH level for DNA binding. The color of our solution was yellow which indicates an optimal pH for DNA binding. But, if the color of solution was orange or violet, 10 µl of 3 M sodium acetate, pH 5.2 solution will be added into the gel mixture for obtaining the yellow color. Also, we’ve used the 100% isopropanol for precipitation of DNA and Wash Buffer for removal of undesired components. Until the elution stage, high salt condition can be provided the attachment of DNA molecules to the GeneJET purification column, but in the elution stage low salt condition can be provided the collection of DNA molecules by Elution Buffer solution for downstream applications. At the end of the gel extraction method, we’ve stored some of purified DNA at -20 oC and used some (2 µl) for Nanodrop measurements. We’ve measured the absorbance values including the A260/A280 and A260/A230 ratios for checking the purities of extracted DNA molecules and also total DNA amount (ng/ µl) by using the Nanodrop Spectrophotometry and recorded the datas which shown table 2. Based on the Nanodrop results, we’ve checked the purities of collected DNA molecules for our group and observed that the extracted DNA molecules were pure when we’ve examined the A260/A280 ratio, because if the A260/A280 ratio is about 1.8, this means the DNA sample is pure, so we’ve not observed any protein or phenol contamination. However; when we’ve look at the A260/A230 ratio, we’ve observed that the alcohol contamination. Because if the is less than 1.8, it can be said that there is alcohol contamination. The reason of this situation can be presence of organic contaminants such as phenol or other organic compounds. Because many organic compounds can show the strong absorbances at about 225 nm causing interfere with downstream applications [8]. Also, we’ve determined the total DNA amount some less as 20.7 ng/ µl than expected result. The reason of this can be incomplete solubilization of the gel slice, inefficient DNA binding, membrane wash or DNA elution. At the end of this experiment, we’ve used this data for the calculation of required DNA amount which will be used for the ligation reaction in TA cloning processes. 

REFERENCES 

  1. PCR: How We Copy DNA 

http://molecular.roche.com/pcr/Pages/Process.aspx 

  1. Polymerase Chain Reaction (PCR) 

http://vlab.amrita.edu/?sub=3&brch=186&sim=321&cnt=1 

  1. Innis, M.A., Myambo, K.B., Gelfand, D.H. and Brow, M.A.D. (1988) Proc. Natl. Acad. Sci. USA 85, 9436. 

Barnes, W.M. (1994) Proc. Natl. Acad. Sci. USA 91, 2216. 

Tindall, K.R. and Kunkel, T.A. (1988) Biochemistry 27, 6008. 

Chou, Q., Russel, M., Birch, D., Raymond, J., Bloch, W. (1992) Nucl. Acids Res., 20, 1717. 

Sharkey, D.J., Scalice, E.R., Christy, K.G., Atwood, S.M., Daiss, J.L. (1994) BioTechnology, 12, 506. 

  1. Apte A., Daniel S. (2009) PCR primer design. Cold Spring Harb. Protoc. doi:10.1101/pdb.ip65. 

Newton C.R., Graham A. (1994) Introduction to biotechniques (Bios Scientific, Oxford, UK). 

  1. Purifying DNA from an Agarose Gel 

https://www.addgene.org/plasmid-protocols/gel-purification/ 

  1. DNA extraction from agarose gels (basic method) 

http://www.methodbook.net/dna/gelextrc.html 

  1. PCR Troubleshooting Guide 

https://www.neb.com/tools-and-resources/troubleshooting-guides/pcr-troubleshooting-guide 

  1. Interpreting Nanodrop (Spectrophotometric) Results 

http://www.u.arizona.edu/~gwatts/azcc/InterpretingSpec.pdf 

Amplification of TAT Gene and Purification by Gel

 AIM 

Aim of this experiment was to amplify TAT gene by PCR technique and to purify the obtained TAT gene fragments by applying the gel extraction method such as Thermo Scientific GeneJET PCR Purification Kit for the TA-cloning of TAT.  

INTRODUCTION 

Polymerase Chain Reaction 

PCR (polymerase chain reaction) is a common laboratory technique which provides the amplification of a specific segment of DNA sequence from a mixture of DNA molecules. The polymerase chain reaction is performed into three stages including the denaturation, annealing and elongation or extension which are also called PCR cycle. In the denaturation stage, applying the heat (more than 90 oC) separates the double-stranded DNA into two single-stranded DNA by breaking down the weak hydrogen bonds between the bases. However; the stronger bonds between deoxyribose and phosphates do not break. In the annealing stage, the DNA mixture is cooled (40 oC and 65 oC) allowing the primer binding and annealing to target complementary sequence in the template DNA. In the elongation stage, the temperature is increased to 72 oC which is optimal temperature for DNA polymerase activity allowing the DNA polymerase to synthesize new strand of DNA by also adding the nucleotides (dNTPs) onto the end of the extending primers. Synthesis of new strand always starts at 3’ end of the primer and continues in the 5’ to 3’ direction. As a result, two new DNA strands identical to original target can be amplified. The PCR cycle is repeated many times (30-40) and by this way, the synthesized products in a given cycle can serve as templates for the next cycles and so the number of target DNA copies become about double in every cycle [1]. 

The PCR reaction is set up with essential components including the a template DNA, a suitable DNA polymerase enzyme like Taq DNA polymerase, a buffer solution for the enzyme, magnesium chloride, dNTP mix, forward and reverse primers and water. By combining of all these components, a reaction mixture is prepared for performing the PCR. In this case, a template DNA also known as obtained DNA from samples includes target sequence of interest which will be amplified.  The DNA polymerase is an enzyme which synthesizes a new strand of DNA also via sequential addition of nucleotides [2]. Taq DNA polymerase is a highly processive thermostable enzyme which synthesized a new strand of DNA by addition of dNTPs onto the end of an annealed primer. For its activity, it needs to magnesium ions in which magnesium chloride. Taq DNA polymerase also adds the single deoxyadenosine (A) to 3’ end of the PCR product which is ligated with 3’ T overhangs of the cloning vector for the TA cloning process [3]. Buffer is a salt-solution which provides the optimal pH and salt condition for DNA polymerase and other reaction components. Primers including the forward and reverse primers are small segments of single stranded DNA (15-30 bases long) which provide annealing to their complementary sequence on the template strand. They provide initiation sites for DNA synthesis by binding to end of the target DNA sequence. In the PCR technique, magnesium as a cofactor is used in the magnesium chloride form and it provides the optimum activity for DNA polymerase enzyme. Lastly, dNTPs are four nucleotides which extend an annealed primer by acting of DNA polymerase [2]. 

For the PCR optimization, each required component must be mixed with appropriate concentrations of components during the PCR reaction. Buffer concentration including the pH and salt concentration must be in an appropriate concentration in which high salt conditions damage the DNA by denaturation resulting in the failure of the reaction and also low pH conditions affect the enzymatic activity (not worked). Also primer concentration must be appropriate at which high primer concentration causes mispriming and accumulation of nonspecific products resulting in decreasing the specificity and at which low concentration causes the completing reaction early resulting in the less yield of desired product. Moreover, high enzyme concentration causes the amplification of non-specific annealed primers resulting in the decreasing the specificity and low enzyme concentration causes low amplification of annealed primers resulting in the less yield of desired product. High DNA concentration causes the false priming resulting in the less DNA synthesis and low DNA concentration causes the less yield of desired product. The last important factor in PCR optimization is water concentration which arranges the optimal pH and salt concentration by dilution of buffer like from 10X to 1X [4].    

Gel Extraction 

Gel extraction is a method allowing the high purity of DNA which is used to isolate and purify the desired fragment of DNA from other bigger and smaller DNA (non-specific) based on their size by cut out of the visualized band on the agarose gel with performing the specific gel extraction processes especially spin-column kit procedure [5][6]. 

 

 

MATERIALS&METHODS 

PCR Reaction for TAT Gene 

For the PCR reaction, the isolated pCV-1 plasmid including the TAT gene from previous experiment was used as a template. The reaction mixture was prepared as 4 reactions for preventing losing of materials in the presence of any problems. Thereby, required amounts of all PCR components waited on ice were mixed (except the DNA) for the four reactions master mixture in order as shown below table:  

Table 1: The required amounts of PCR components for one reaction and four reactions to set up PCR reaction mixtures 

Component  Volume (µl)  Master Mix Volume (4 rxn) 
Nuclease-free dH2O  13.5 µl  54 µl 
10X Taq Buffer  2.5 µl  10 µl 
dNTP mix, 10 mM each  1 µl  4 µl 
25 mM MgCl2  2.5 µl  10 µl 
Forward primer (2 pmol)  2 µl  8 µl 
Reverse Primer (2 pmol)  2 µl  8 µl 
Taq Polymerase (5u/µl)  0.5 µl  2 µl 
DNA ng/µl  1 µl  Total 24 µl for each tube 

+ 1 µl of DNA 

TOTAL  25 µl 

 

After set up of master mix, 24 µl of mixture without the DNA sample was transferred into 2 PCR tubes for each group and then 1 µl of DNA was added into the each tube as the total volume of reaction mixture has been reached to 25 µl. The PCR tubes holded on ice was gently flicked to mix the solution and briefly centrifuged. Finally, the PCR tubes were placed into PCR machine and PCR cycles were performed under certain conditions as below: 

 

  • Initial Denaturation: 94 oC for 3 min 
  • Denaturation: 94 oC for 30 sec 
  • Annealing: 53 oC for 30 sec            30 cycle 
  • Extension: 72 oC for 45 sec 
  • Final Extension: 72 oC for 5 min 

Agarose Gel Electrophoresis 

After performing the PCR reaction, the PCR products including the amplified TAT gene and other molecules were loaded into an agarose gel. Before loading the samples, 0.8% agarose gel was prepared previously. In this case, to prepare 8% agarose gel, 1.2 g agarose was weighed and it was solved into 150 ml TAE Buffer by applying the heat and then cooled for addition of intercalating agent ethidium bromide. After preparation of agarose gel, all of 25 µl of PCR products were loaded into three gel wells with mixing 5 µl of 6X loading dye as the last concentration of loading dye comes to 1X which calculation for it is shown as below: 

Stock concentration of loading dye = 6X 

Final concentration of loading dye = 1X     So; 

M× V1 = M× V2 

1X × (25 µl +?) = ? × 6X                    

? = 5 µl of loading dye required 

The gel was run by 100 V electric current for 30 min. Finally, the gel slice including the desired DNA fragments were removed from the agarose gel by cut out of them under the UV light and the removed gel slices (2 gel slices for each group) were weighed for the next processes as gel extraction. 

Gel Extraction  

Firstly, the weight of cut out gel slices containing the fragment were determined as 0.2157 g ~ 216 mg for our group and 0.1666 g ~ 167 mg for other subgroup. The gel extraction processes were performed by Thermo Scientific GeneJET PCR Purification Kit. Then 1:1 volume of Binding Buffer, 216 µl of Binding Buffer was added into the gel slice and the gel mixture was incubated at 60 oC for 10 min until the gel slice was completely dissolved. In this stage, the tube with gel slice was mixed by inversion at certain intervals to facilitate the melting of gel slice. On the other hand, during dissolving, the color of solution was checked to control pH level for DNA binding. The color of our solution was yellow which indicates an optimal pH for DNA binding. But, if the color of solution was orange or violet, 10 µl of 3 M sodium acetate, pH 5.2 solution will be added into the gel mixture for obtaining the yellow color. After the gel slice completely dissolved, a 1:2 volume of 100% isopropanol, 216 µl of 100% isopropanol was added into solubilized gel solution and it was mixed. Then, up to 800 µl of the solubilized gel solution was transferred to the GeneJET purification column and centrifuged for 1 min. After the centrifugation, the supernatant was removed and the column was placed back into the same collection tube. After that, 700 µl of Wash Buffer added into the GeneJET purification column and centrifuged for 1 min and again the supernatant was removed. The empty GeneJET purification column was centrifuged for an additional 1 min to completely removal of residual wash buffer. Then, the GeneJET purification column of one subgroup was transferred into a clean 1.5 ml microcentrifuge tube and 45 µl of Elution Buffer was added to the center of the purification column membrane and centrifuged for 1 min. After centrifugation, the same Elution Buffer (45 µl) was used for our group’s GeneJET purification column and again centrifuged for 1 min. Finally, the purified DNA of each subgroup of our group was collected into the same tube by discarding the GeneJET purification columns and some of purified DNA was stored at -20 oC and some (2 µl) was used for Nanodrop measurements. 

Nanodrop Measurements 

For the Nanodrop measurements, firstly, 2 µl of dH2O was put into the pit of Nanodrop machine for cleaning the pit and it was dried. Then 2 µl of solvent of DNA (TE/ dH2O) was put into the pit as a blank in order to calibrate and pit was dried again. 2 µl of sample (DNA in TE/dH2O) was put into the pit and absorbance values including the A260/A280 and A260/A230 and also total DNA amount (ng/ µl) were determined and the results were recorded. 

 

RESULTS 

By performing the polymerase chain reaction we’ve amplified the TAT gene and also we’ve visualized the TAT gene fragments for each group by applying the agarose gel electrophoresis method and so, we’ve examined the PCR products on the agarose gel under the UV light which shown in below figure; 

   

Figure 1: The agarose gel image of amplified TAT genes results of the performing the PCR reaction in the presence of a ladder with 1 kb length bond size. TAT gene has a 263 bp length and so, the bands at approximately 300 bp indicate the amplified TAT genes. In addition to this, G1-1, G1-2 and G1-3 DNA bands indicate that the 1st group’s amplified PCR products including TAT gene with approximately 300 bp. G2-1, G2-2 and G2-3 DNA bands indicate that the 2nd group’s amplified PCR products including TAT gene with approximately 300 bp. G3-1, G3-2 and G3-3 DNA bands indicate that the 3rd group’s amplified PCR products including TAT gene with approximately 300 bp which not work the PCR correctly. Because, the bands are not very clear on the gel image especially the indicated band of G3-3 as shown above figure. G4-1, G4-2 and G4-3 DNA bands indicate that the 4th group’s amplified PCR products including TAT gene with approximately 300 bp. G5-1, G5-2 and G5-3 DNA bands indicate that the 5th group’s amplified PCR products which not worked PCR and so we’ve not observed any bands for TAT gene with approximately 300 bp in the 5th group’s gel results. 

After the agarose gel electrophoresis each group has cut out the own gel slice from the agarose gel under the UV light and performed the gel extraction method to isolate and purify the desired fragment of DNA from other bigger and smaller DNA molecules (non-specific). Thereby, at the end of the gel extraction processes, we’ve measured the absorbance values including the A260/A280 and A260/A230 ratios for checking the purities of extracted DNA molecules and also total DNA amount (ng/ µl) by using the Nanodrop Spectrophotometry and recorded the datas which shown below table: 

 

Table 2: The measured results of purified DNA molecules including the TAT gene with absorbance ratios and also total amount after by performing the gel extraction processes  

  Gel Extraction Method 
A260/A280  1.88 
A260/A230  0.05 
Total DNA (ng/µl)  20.7 ng/µl 

 

Based on the Nanodrop results, we’ve checked the purities of collected DNA molecules for our group and observed that the extracted DNA molecules were pure when we’ve examined the A260/A280 ratio, whereas when we’ve look at the A260/A230 ratio, we’ve observed that the alcohol contamination. Also, we’ve determined the total DNA amount as 20.7 ng/ µl which was unexpected result. We’ve used this data for the calculation of required DNA amount which will be used for the ligation reaction. 

DISCUSSION 

In this lab session, we’ve aimed the amplification of TAT gene by performing the polymerase chain reaction in the presence of required reaction components and we’ve aimed the controlling of amplified TAT genes by agarose gel electrophoresis method followed by the cut out of the gel slice with desired DNA fragment, we’ve aimed to purify the DNA molecules with amplified TAT gene fragments by applying the gel extraction method such Thermo Scientific GeneJET PCR Purification. For this purposes, at first we’ve performed the polymerase chain reaction by preparation of master mix including four PCR reaction mixtures for preventing losing of materials in the presence of any problem such as in pipetting, mixing, etc. In the PCR reaction, we’ve used the isolated pCV-1 plasmid from previous experiment as a template which includes target sequence of interest as TAT gene. Moreover, we’ve used the essential amounts of other PCR components such as MgCl2, dNTP mix, forward and reverse primers, Taq DNA polymerase enzyme and buffer for Taq DNA polymerase as shown in table 1. We’ve used the Taq DNA polymerase for synthesizing the new strand of DNA with also sequential addition of nucleotides like dNTPs. Also we’ve used it for production of PCR product with 3’ end which is necessary for TA cloning processes. The Taq DNA polymerase activity depends on the magnesium ions and buffer conditions. Magnesium ions interact with negatively charged molecules due to its positive charge in the reaction such as template DNA, primers and also dNTPs [2]. Moreover, we’ve used the buffer solution for optimal pH and salt condition for DNA polymerase activity and stabilization of other components. Also we’ve used the forward and reverse primers extended by dNTPs for annealing to their complementary sequences on the template strand. When we’ve set up the PCR reaction, we’ve mixed the PCR components in an order as water, buffer, dNTP mix, magnesium chloride, forward and reverse primers, Taq DNA polymerase and lastly template DNA. We’ve mixed the water and buffer at first because the water decreases the harmful effect of buffer solution on enzyme activity and other components by diluting the buffer. We’ve added the enzyme and DNA lastly for preventing the any damage to enzyme or starting reaction from nonspecific regions at which primers bind randomly. At the end of this stage, we’ve total 25 µl of reaction mixture and performed the PCR cycles under the suitable PCR conditions. After performing the PCR reaction, we’ve prepared the 0.8% agarose gel which 1.2 g of agarose was solved in 150 ml TAE buffer by applying the heat and then the solution cooled before addition of ethidium bromide intercalating agent for preventing the its harmful effect. We’ve prepared the 0.8% agarose gel for the separation of large DNA fragments. So, we’ve loaded the all of PCR products into three gel wells with mixing 6X loading dye as the last concentration of loading dye as 1X. We’ve used the loading dye including the glycerol and bromophenol blue to sinking of the samples into the well by glycerol and visualization of DNA migration by bromophenol blue. Also we’ve used the ethidium bromide intercalating agent for visualization of DNA bands under the UV light. After the agarose gel electrophoresis, we’ve cut out the gel slices (two gel slices for each group) for the gel extraction processes. Based on the agarose gel image shown in figure 1, we’ve expected to observe bands at 263 bp (~ 300 bp) length for amplified TAT genes. In addition to this, we’ve observed the approximately 300 bp bands for the G1-1, G1-2 and G1-3 DNA bands which indicate the 1st group’s amplified PCR products and this result was expected. We’ve observed the approximately 300 bp bands for the G2-1, G2-2 and G2-3 DNA bands which indicate the 2nd group’s amplified PCR products. But the band in G2-1 was clearer than G2-2 and G2-3. The reason of these less clear bands can be excess amount of enzyme and long extension time [8]. We’ve observed the approximately 300 bp bands for the G3-1, G3-2 and G3-3 DNA bands which indicate the 3rd group’s amplified PCR products. But the band in G3-2 was clearer than G3-1 and G3-3. The reason of these less clear bands can be excess amount of enzyme and long extension time [8]. We’ve observed the approximately 300 bp bands for the G4-1, G4-2 and G4-3 DNA bands which indicate the 4th group’s amplified PCR products and this result was expected. Moreover, we’ve not observed any bands for G5-1, G5-2 and G5-3 DNA bands which indicate that the 5th group’s amplified PCR products. This means that the PCR cycles have not performed correctly or the agarose gel electrophoresis has not performed correctly even TAT gene fragments have amplified. This result was unexpected. The reason of this can be too short extension time, very high annealing temperature, and inappropriate buffer causing the enzyme or DNA denaturation, low primer concentration, long denaturation time and inappropriate amount of magnesium ions [7]. After the agarose gel electrophoresis, we’ve performed the gel extraction method by using the Thermo Scientific GeneJET PCR Purification Kit. We’ve performed the gel extraction method because, the PCR mixture was not used directly in cloning and in addition to this and to remove the other bigger and smaller nonspecific DNA molecules and we’ve isolated and purified the desired fragment of DNA. For this purposes, at first we’ve determined the weights of the gel slices containing the fragment as 0.2157 g ~ 216 mg for our group and 0.1666 g ~ 167 mg for other subgroup. Based on these weights we’ve performed other processes. We’ve used Binding Buffer for dissolving the gel slice by incubation at 60 oC. In this case, the concentration of gel slice was depending on the buffer, heat and incubation time. On the other hand, during dissolving, we’ve checked the color of solution to control pH level for DNA binding. The color of our solution was yellow which indicates an optimal pH for DNA binding. But, if the color of solution was orange or violet, 10 µl of 3 M sodium acetate, pH 5.2 solution will be added into the gel mixture for obtaining the yellow color. Also, we’ve used the 100% isopropanol for precipitation of DNA and Wash Buffer for removal of undesired components. Until the elution stage, high salt condition can be provided the attachment of DNA molecules to the GeneJET purification column, but in the elution stage low salt condition can be provided the collection of DNA molecules by Elution Buffer solution for downstream applications. At the end of the gel extraction method, we’ve stored some of purified DNA at -20 oC and used some (2 µl) for Nanodrop measurements. We’ve measured the absorbance values including the A260/A280 and A260/A230 ratios for checking the purities of extracted DNA molecules and also total DNA amount (ng/ µl) by using the Nanodrop Spectrophotometry and recorded the datas which shown table 2. Based on the Nanodrop results, we’ve checked the purities of collected DNA molecules for our group and observed that the extracted DNA molecules were pure when we’ve examined the A260/A280 ratio, because if the A260/A280 ratio is about 1.8, this means the DNA sample is pure, so we’ve not observed any protein or phenol contamination. However; when we’ve look at the A260/A230 ratio, we’ve observed that the alcohol contamination. Because if the is less than 1.8, it can be said that there is alcohol contamination. The reason of this situation can be presence of organic contaminants such as phenol or other organic compounds. Because many organic compounds can show the strong absorbances at about 225 nm causing interfere with downstream applications [8]. Also, we’ve determined the total DNA amount some less as 20.7 ng/ µl than expected result. The reason of this can be incomplete solubilization of the gel slice, inefficient DNA binding, membrane wash or DNA elution. At the end of this experiment, we’ve used this data for the calculation of required DNA amount which will be used for the ligation reaction in TA cloning processes. 

REFERENCES 

  1. PCR: How We Copy DNA 

http://molecular.roche.com/pcr/Pages/Process.aspx 

  1. Polymerase Chain Reaction (PCR) 

http://vlab.amrita.edu/?sub=3&brch=186&sim=321&cnt=1 

  1. Innis, M.A., Myambo, K.B., Gelfand, D.H. and Brow, M.A.D. (1988) Proc. Natl. Acad. Sci. USA 85, 9436. 

Barnes, W.M. (1994) Proc. Natl. Acad. Sci. USA 91, 2216. 

Tindall, K.R. and Kunkel, T.A. (1988) Biochemistry 27, 6008. 

Chou, Q., Russel, M., Birch, D., Raymond, J., Bloch, W. (1992) Nucl. Acids Res., 20, 1717. 

Sharkey, D.J., Scalice, E.R., Christy, K.G., Atwood, S.M., Daiss, J.L. (1994) BioTechnology, 12, 506. 

  1. Apte A., Daniel S. (2009) PCR primer design. Cold Spring Harb. Protoc. doi:10.1101/pdb.ip65. 

Newton C.R., Graham A. (1994) Introduction to biotechniques (Bios Scientific, Oxford, UK). 

  1. Purifying DNA from an Agarose Gel 

https://www.addgene.org/plasmid-protocols/gel-purification/ 

  1. DNA extraction from agarose gels (basic method) 

http://www.methodbook.net/dna/gelextrc.html 

  1. PCR Troubleshooting Guide 

https://www.neb.com/tools-and-resources/troubleshooting-guides/pcr-troubleshooting-guide 

  1. Interpreting Nanodrop (Spectrophotometric) Results 

http://www.u.arizona.edu/~gwatts/azcc/InterpretingSpec.pdf 

Isolation of PCV-1 and Confirmation by Restriction Digestion

AIM 

Aim of this experiment is the isolation of pCV1 plasmid DNA from E.coli bacterial culture grown overnight in LB medium with a suitable antibiotic such as tetracycline which selects the cells having plasmid and so tetracycline resistance gene. Also, aim is to cut the pCV1 plasmid by PstI restriction enzyme for controlling that isolated plasmid is actually pCV1 or not via Nanodrop measurements and also agarose gel electrophoresis. 

INTRODUCTION 

Plasmid is an extra chromosomal, supercoiled, circular double stranded DNA molecule which found in bacteria and some eukaryotes. Plasmids have major three elements including an origin of replication, an antibiotic resistance gene like resistance to ampicillin and also a multiple cloning site. Moreover, they have selectable markers, promoter region and primer binding site. Plasmids provide the adaptation to the host cells under the unavailable environmental conditions via their included genes [1]. Plasmids can be important and essential for many cellular processes in molecular biology such as determination of the gene function, because they are easy to work due to their stabilities and capable of independent replication [2]. pCV1 plasmid derived from pCV is a mammalian expression vector which includes  the hybrid regulatory sequences of HIV and also tetracycline resistance gene. This plasmid can be cut by PstI restriction enzyme which forms two fragments like 3.3 kb and 7.0 kb [3]. 

Isolation of Plasmid DNA  

Plasmid DNA isolation is a process in which plasmid DNA is obtained by separating from other cellular components via applying physical or chemical methods. In the plasmid isolation, there are 4 basic stages. Firstly, a bacterial culture is grown overnight in a LB medium with a suitable antibiotic. Generally, ampicillin and kanamycin are used for selection. After that, obtained bacterial culture containing plasmid of interest is harvested by spinning and lysate by physical or chemical methods such as using of the chemical agents that break down the cell integrity. Lysozyme, EDTA, SDS and strong alkali reagent are examples of these chemical agents. Lysozyme breaks down the cell wall polymeric compounds which provide the integrity. EDTA damages the cell envelope structure by removal of magnesium ions from the cell membrane. SDS is a detergent which removes the lipid molecules from cell membrane in the lysis stage. Also, strong alkali reagents denature the host cell chromosomal DNA without any damaging the plasmid DNA due to its compact structure. After the lysis of the cells, all components are removed except the plasmid DNA and finally, plasmid DNA is purified [4]. For the plasmid DNA isolation, many methods are used including the Miniprep Kit and Alkaline Lysis methods. ALS is a method which isolates the plasmid DNA in the presence of three buffer solutions with different chemicals which are glucose, Tris-HCl and EDTA for Alkaline Lysis Buffer I; NaOH and SDS for Alkaline Lysis Buffer II; potassium acetate and glacial acetic acid for Alkaline Lysis Buffer III.  

Miniprep method also defined as minipreparation of plasmid DNA depends on the small-scale for the isolation of plasmid DNA from bacteria by usage of a spin-column and also different types of chemicals. 

DNA Concentration Determination 

In the molecular biology, nucleic acid quantitation is important that how much DNA or RNA is present in the prepared solutions for performing the experiments about the genes. So, DNA concentration in a sample is determined by UV absorbance spectrophotometry at 260 nm. The UV absorbance spectrophotometry can also assess the purity of a DNA sample based on the ratio of absorbance at 260 and 280 nm. For the purity of a DNA sample, the A260/A280 ratio is accepted as 1.8. However; if this ratio become less than 1.8, this means that there is any protein or phenol contamination, because proteins can give absorbance at 280 nm. If the ratio become above 1.9, this means that there is RNA contamination, because RNA can give absorbance at 260 nm. Moreover, A260/A230 ratio is used for determination of purity which indicates the alcohol contamination of any solutions, when the ratio becomes less than the 1.8 [5]. 

Restriction Enzyme 

Restriction enzymes also called restriction endonucleases are DNA-cutting enzymes in the bacteria which cleave DNA at the specific recognition sequences, palindromic sequences, by recognizing them for producing the 5’ phosphates and 3’ hydroxyls. A palindromic sequence is the same nucleic acid sequence when it read from 5’ to 3’ or 3’ to 5’. Restriction enzymes naturally can be found in bacteria types. Bacteria use them for protection themselves against the phage DNA and they have ability to prevent them cut by their restriction enzymes. They can change the recognition sequence of the enzyme and so, the enzyme does not work. Also, restriction enzymes form 2 types of ends including sticky-cohesive ends or blunt ends. Sticky-cohesive ends include 5’ or 3’ overhangs due to asymmetrically cutting of the enzyme such as Bam HI (5’ overhangs) and Kpn I (3’ overhangs). In contrast, the blunt ends occur without any overhangs, when the enzyme like Sma I cut the both strand of DNA in the middle of the recognition sequence [6]. 

Star Activity 

Under the extreme reaction condition in terms of elevated pH or low ionic strength, restriction enzymes can cleave similar, non-identical sequences and this condition can change the specificity resulting in the star effect or star activity [7]. 

Agarose Gel Electrophoresis 

Agarose gel electrophoresis is a method that separates the molecules based on their molecular weights through a gel by applying an electric field. The movement of molecules is from the cathode (-) towards the anode (+) due to negative charge of phosphate group in the double-stranded DNA molecules at neutral pH. The supercoiled DNA moves faster than the linear form of the same plasmid and linear DNA moves faster than the circular nicked form of the same plasmid. Moreover, the movement of the DNA molecules can be affected by other factors including the conformation of nucleic acids, buffer composition, agarose concentration, strength of electric field and excess amount of intercalating dye such as ethidium bromide. In the agarose gel electrophoresis procedure, the samples are loaded into the wells by mixing DNA samples with a loading dye which includes glycerol and bromophenol blue. In this case, glycerol provides the samples to sink into the wells and bromophenol blue provides to visualize the DNA migration through the agarose gel. Also ethidium bromide provides to visualize the DNA bands in the agarose gel under the UV light [8]. 

MATERIALS&METHODS  

 pCV-1 plasmid                               –Ethidium bromide                          –Eppendorf tubes 

–Nanodrop spectrophotometry         –Restriction enzyme PstI                 –Pipette and tips 

–LB medium                                     –6X Gel loading dye                        –Glove 

–Tetracycline                                    –Miniprep Kit                                   —E.coli bacteria 

–TAE buffer                                     –Agarose gel electrophoresis apparatus 

–Alkaline Lysis Buffer I                  –Alkaline Lysis Buffer III 

–Alkaline Lysis Buffer II                 –Ethanol (100%-70%) 

ALS METHOD 

Firstly, overnight E.coli culture including the pCV-1 plasmid and TAT gene was centrifuged at ≥12000 x g for 1 min and supernatant was discarded. Then, bacterial culture was added into the remained pellet and centrifuged at ≥12000 x g for 1 min again for the precipitation of bacterial cells and the supernatant was discarded again. After that, 100 µl of ice-cold Alkaline Lysis Solution I is added into the remained pellet and gently vortexed. Then, 200 µl of Alkaline Lysis Solution II is added into the mixed solution without vortexing for preventing of plasmid and then 150 µl of Alkaline Lysis Solution III was added into the solution and waited on the ice for 3 minutes. After 3 min, the bacterial lysate was centrifuged at maximum speed for 5 minutes at +4 oC. After the centrifugation, the supernatant was transferred into a new Eppendorf tube and then 900 µl of 100% ethanol was added into the solution to precipitate the nucleic acids and vortexed and waited for 2 minutes at room temperature. Then, precipitated nucleic acids were centrifuged at maximum speed for 5 minutes at +4 oC in a microfuge for the collection of nucleic acids. After centrifugation, the supernatant was discarded and remained pellet was solved with 1 ml of 70% ethanol by inverting of the closed tube several times and again centrifuged at maximum speed for 2 minutes at +4 oC in a microfuge for recovering of the DNA. After centrifugation, the supernatant was discarded gently and the open tube was waited for evaporation of all liquid. Finally, the plasmid DNA was collected in the eluate and the amount of DNA was measured by Nanodrop spectrophotometry. 

Restriction reaction for ALS; 

After the Nanodrop measurements, the required amounts of buffer O (10X), plasmid DNA and water were calculated as 2 µl, 0,95 ~ 1 µl and 16 µl, respectively for the restriction reaction. Firstly, water and buffer were mixed and then plasmid DNA was added into the reaction mixture and finally, alredy calculated 1 µl of PstI enzyme was added into the mixture and mixture was incubated at 37 oC for 1 hour. 

MINIPREP KIT METHOD 

All stages in the kit were performed at room temperature. Firstly, overnight E.coli culture including the pCV-1 plasmid and TAT gene was centrifuged at ≥12000 x g for 1 min for harvesting of the cells. After the centrifugation, supernatant was removed and 1 ml of cells were added into the pellet in the microcentrifuge tube and then again it was centrifuged at ≥12000 x g for 1 min and after the centrifugation the supernatant was removed. After that, the remained bacterial pellet was resuspended with 200 µl of the Resuspension Solution including the RNase A by applying the pipetting until completely resuspension. Then, 200 µl of Lysis Buffer was added into the resuspended cells for the lysis of bacterial cells and waited for 5 minutes. After 5 minutes, 350 µl of Neutralization/Binding Buffer was added into the cells by inverting it 4-6 times and then the solution was centrifuged to precipitate the cell debris at max speed for 10 min. At the same time, column preparation was made by adding the 500 µl of Column Preparation Solution into a GenElute HP Miniprep Binding Column and applied the vacuum until the whole solution passes through the column. After that, the lysate formed in neutralization stage was transferred into the column and centrifuged at ≥12000 x g for 1 min and the passed lysate through the column was removed. Then, column was washed with 500 µl of Wash Solution 1 and again the passed solution through the column was removed and again column was washed with 750 µl of diluted Wash Solution 2 and passed solution through the column was removed. After this stage, the column was transferred into a new microcentrifuge tube (2 ml) and centrifuged at ≥12000 x g for 1 min. Then 30 µl of Elution Solution was added into the column for obtaining only plasmid DNA. Finally, the plasmid DNA was collected in the eluate and the amount of DNA was measured by Nanodrop spectrophotometry. 

Restriction reaction for Miniprep; 

After the Nanodrop measurements, the required amounts of buffer O (10X), plasmid DNA and water were calculated as 2 µl, 4.36 ~ 4.5 µl and 12.5 µl, respectively for the restriction reaction. Firstly, water and buffer were mixed and then plasmid DNA was added into the reaction mixture and finally, alredy calculated 1 µl of PstI enzyme was added into the mixture and mixture was incubated at 37 oC for 1 hour. 

RESULTS 

Results of Nanodrop Spectrophometry 

At the end of the plasmid DNA isolation processes, we’ve used the plasmid DNAs eluted from both Miniprep Kit and ALS methods for measurement of the total DNA amount in the samples which also required for the restriction reactions and also determining the ratio of A260/A280 and A260/A230 to check the purities of the obtained DNA samples. Thereby, we’ve placed the 2 µl of DNA samples into the Nanodrop spectrophotometry and measured the each value which given below table 1: 

Table 1: indicates that Nanodrop spectrophotometry results for plasmid DNA samples obtained by ALS and Miniprep Kit method 

  Miniprep Kit Method  ALS Method 
A260/A280  1.77  1.68 
A260/A230  1.81  1.83 
Total DNA (ng/µl)  229.2  5248.9 

 

Based on the determined total DNA amount for both Miniprep Kit and ALS method, we’ve calculated the required DNA amount for the restriction reactions as the total volume of reaction mixture has been reached to 20 µl. So, we’ve calculated the required DNA amount as 4.36 µl ~ 4.5 µl for restriction reaction of Miniprep Kit and as 0.19 µl ~ 0.2 µl for restriction reaction of ALS method which are shown below: 

Calculations for Miniprep Kit:           1µl             229.2 ng DNA  

x               1000 ng 

x = 1000/ 229.2 = 4.36 µl plasmid DNA 

We’ve calculated the required amount of DNA as 4.36 µl by using above equation but, we’ve used 4.5 µl of DNA in the restriction reaction and for the complete reaction we’ve calculated the amount of buffer and water which will be used in restriction reaction as shown below: 

M1 × V1 = M2 × V2                                So,       4.5+2+1 = 7.5 µl  

10X × V1 = 1X × 20 µl                                      20 µl – 7.5 µl = 12.5 µl of water 

V= 2 µl of buffer                                             (1 µl is the amount of PstI enzyme) 

 

Calculations for ALS:                       1µl              5248.9 ng DNA  

x               5000 ng 

x = 5000/5248.9 = 0.95 µl plasmid DNA 

We’ve calculated the required amount of DNA as 0.95 µl by using above equation but, we’ve used 1 µl of DNA in the restriction reaction and for the complete reaction we’ve calculated the amount of buffer and water which will be used in restriction reaction as shown below: 

M1 × V1 = M2 × V2                                            So,     1+2+1 = 4 µl  

10X × V1 = 1X × 20 µl                                      20 µl – 4 µl = 16 µl of water 

V= 2 µl of buffer                                             (1 µl is the amount of PstI enzyme) 

 

DISCUSSION 

In this experiment, at first, we’ve aimed the isolation of plasmid DNA including TAT gene from overnight E.coli bacterial culture in the LB medium including the tetracycline and so we’ve eliminated some of bacteria which are sensitive against tetracycline antibiotic, in other words, not have plasmid. Because, the pCV1 plasmid contains the tetracycline resistance gene and so we’ve selected the bacterial cells without plasmid by this way. Also, we’ve aimed to check that isolated plasmids are actually pCV1 or not in the presence of PstI restriction enzyme. For the isolation of plasmid DNA, we’ve used the 2 different methods including Miniprep Kit and ALS methods. In the Alkaline Lysis method, we’ve used some chemicals in which Alkaline Lysis Buffer I, II and III. Firstly, we’ve used the Alkaline Lysis Buffer I containing the glucose, EDTA and Tris-HCl agents for resuspension stage of isolation procedure. Glucose maintains the osmotic pressure for preventing the cells to burst. EDTA is chelating agent that discards the Mg ions which important for cell envelope structure and also prevents the activity of DNases which degrade the DNA structure and makes cells more permeable to SDS agent for lysis processes. Therefore, we’ve aimed to preserve the plasmid DNA structure. Also Tris-HCl arranges the pH level of the environment. Secondly, we’ve used the Alkaline Lysis Buffer II containing NaOH and SDS for cell lysis stage of isolation procedure. In this case, NaOH helps to break down the cell wall and also breaks down the hydrogen bonding between the DNA bases, in other words, it makes from double-stranded DNA molecule to single-stranded DNA molecule. SDS is a detergent which provides the solubilization of cell membranes by removal of the lipid molecules and so, it provides the chromosomal DNA and plasmid DNA to pass into the solution. Lastly, we’ve used the Alkaline Lysis Buffer III containing the potassium acetate, glacial acetic acid and water for neutralization and reducing of the pH processes. Potassium acetate provides the reducing the pH level by precipitation of SDS with its lipids and proteins and also glacial acetic acid brings the pH to neutral and so, DNA strands can be renatured. Moreover, at the end of the Alkaline Lysis method, we’ve also used the 100% ethanol to prevent the dissolving of DNA molecule and also to precipitate the other salts or anything and before measurement by Nanodrop, we’ve used 70% ethanol for precipitation of plasmid DNA. After additional of ethanol chemicals, we’ve waited for evaporation of entire alcohol from the DNA sample to obtain accurate measurements by Nanodrop spectrophotometry. Thereby, after evaporation,  we’ve measured the amount of DNA and also A260/A280  and A260/A230 ratios by Nanodrop spectrophotometry. Other method which we’ve used was Miniprep Kit method. Actually, when we’ve compared the two plasmid DNA isolation methods, we’ve realized that Alkaline Lysis is a manual method which used basic three buffers with different components and also alcohol for the precipitation of DNA and so it’s also known as alcohol precipitation method, however; although the Miniprep Kit method is similar to ALS method, it is applied based on the small-scale commercial kit procedures including a spin-column and also different types of solutions and buffers which also show the similar effects with buffer components in ALS method. Also, we can obtain more plasmid DNA molecule by Alkaline Lysis method compared to Miniprep Kit. So, we’ve used the some chemicals including the Resuspension Solution containing RNase, Lysis Buffer, Neutralization/Binding Buffer, a GenElute HP Miniprep Binding Column, Column Preparation Solution, Wash Solution 1 and Wash Solution 2 and Elution Solution during the Miniprep Kit procedure. We’ve used the Resuspension Solution for resuspending the cells like ALS I buffer and in this method it contains the RNase, unlike in the ALS method, there is not RNase. This means, the RNA contamination can observe in the Nanodrop results for ALS method. Also, we’ve used Lysis Buffer for cell lysis stage like ALS II buffer, Neutralization/Binding Buffer for neutralization like ALS III buffer, a GenElute HP Miniprep Binding Column, Column Preparation Solution, Wash Solution 1 and Wash Solution 2 for removal of other components like salts and Elution Solution for elution of plasmid DNA molecules together. At the end of the Miniprep Kit procedure, we’ve measured the amount of DNA and also A260/A280 and A260/A230 ratios by Nanodrop spectrophotometry. According to Nanodrop results, we’ve not observed any alcohol contamination for both ALS and Miniprep Kit. Because, A260/A230 ratio checks the alcohol contamination and we’ve measured the ratio as 1.81 for Miniprep Kit and 1.83 for ALS method. If these values could be less than 1.8, it could be said that alcohol contamination occurred in these procedures. This result was expected. Also we’ve observed protein contamination for both ALS and Miniprep Kit. Because, A260/A280 ratio checks the purity of DNA preparation as 1.8 value and so, we’ve measured both value as less than 1.8 and there can be protein contamination for both procedure. Because, proteins give absorbance at 280 nm and so accumulation of proteins in the samples or not completely removal of them can be caused thid situation. In other words, in the presence of high concentrations of proteins, proteins can contribute to the 260 and 280 absorbance. This result was unexpected. However; when we’ve compared the total DNA amount of both ALS and Miniprep Kit methods, the results were expected meaning that more DNA can obtain by ALS method instead Miniprep Kit method. After the Nanodrop measurements, we’ve prepared the restriction reaction mixture by using the calculated amounts of buffer O (10X), obtained plasmid DNA, PstI restriction enzyme and distilled water. When we’ve prepared the reaction mixture, we’ve take car about the order of materials which will be added and so, we’ve mixed at first distilled water, buffer and plasmid and at last we’ve added the enzyme into the mixture for preventing the enzyme activity before formation of the best media and also preventing the denaturation of enzyme. On the other hand, for the visualizing the uncut and digested DNA molecules obtained by ALS and Miniprep Kit method, we’ve performed the agarose gel electrophoresis method by using the Ethidium bromide for observation of DNA bands under the UV light and loading dye including glycerol providing the samples to sink into the wells and bromophenol blue for visualization of DNA migration through agarose gel. For this process, 1% agarose was prepared in the TAE buffer and samples were run on 100 volt for 30 minutes. For this purpose, the ladder with 10.0 kb in length was used and at the end, the samples were loaded into the wells of gel in order to, first ladder, uncut ALS isolated plasmid (1 µl of plasmid), digested ALS isolated plasmid (5 µg of plasmid), uncut Miniprep isolated plasmid (1 µl of plasmid) and then digested Miniprep isolated plasmid (1µg of plasmid). Thereby, the gel image was obtained. The used pCV1 plasmid is 10.3 kb in length and so when it was digested by enzyme, the digest fragments should be 7.0 and 3.3 kb in length. In other words, we should observe one band at only about 10 kb for uncut ALS and Miniprep isolated plasmid and also two bands at about 7.0 kb and 3.0 kb for digest ALS and Miniprep isolated plasmid. So, depending on the agarose gel image, the results for the digested ALS and Miniprep isolated plasmid were expected. Because, two bands formed at about 7.0 kb and 3.0 kb, but also a smear formed in the lane of digested ALS isolated plasmid which not expected result. The reason of this can be contamination of DNA with protein meaning that can be accumulation of protein or not completely removal of the protein from the samples and also some RNA contamination due to absence of RNase enzyme in the ALS procedure. Also the reason can be excess salts which accumulate by deficiency of ethanol precipitation. Moreover, the DNA can be degraded by DNases due to error in the EDTA agent. However; when we’ve examined the results of uncut ALS and Miniprep isolated plasmids, we’ve observed that more than one band as a smear between 6.0 kb and 10.0 kb for uncut ALS plasmid with also a smear which can be any protein or RNA contamination and also one band at about 8.0 kb and one band at about more than 10 kb for uncut Miniprep plasmid. Actually, we should observe one band for these uncut samples, however; the reason of this can be supercoiled DNA and the reason of other unexpected results can be related to E.coli cells or not completely elution of plasmid. As a result, when we’ve examined the whole gel image, we can also realize that the supercoiled (uncut) DNA migrates faster than linear (digested) form of the same plasmid which also expected result. 

REFERENCES 

  1. Bacterial Plasmids 

https://www.csun.edu/~hcbio027/biotechnology/lec2/PL/pl.htm 

  1. What is a Plasmid? 

https://www.addgene.org/mol-bio-reference/plasmid-background/ 

  1. Arya SK., Guo C, Josephs SF, Wong-Staal, F. Trans-activator gene of human T-lymphotropic virus type III (HTLV-III). Science 229:69-73, 1985. 
  1. Sabine Ehrt and Dirk Schnappinger, Isolation of Plasmids from E. coli by alkaline Lysis, Methods in Molecular Biology, Pages 79-82. 

Craig Winstanley and Ralph Rapley, Extraction and Purification of Plasmid DNA, The Nucleic Acid Protocols Handbook (2000),Pages 327-331 

  1. How do I determine the concentration, yield and purity of a DNA sample? 

https://worldwide.promega.com/resources/pubhub/enotes/how-do-i-determine-the-concentration-yield-and-purity-of-a-dna-sample/ 

  1. REBase: Database from New England Biolabs with detailed information on restriction endonucleases. 
  1. Nasri, M. and Thomas, D. (1986) Nucleic Acids Res. 14, 811. PMID: 3003698 
  1. Gel Electrophoresis 

http://www.phschool.com/science/biology_place/labbench/lab6/gelelect.html 

 

 

 

Best model organisms for chromatin studies

The first model organism whose chromatin I would start study is the Tetrahymena thermophila that satisifies the basic conditions of a good model organism, such as short life cycle, low cost, accesibility to both forward and reverse genetics. Furthermore being a low eukaryote (unicellular), it is an excellent model for studying the chromatin from both the evolutionary, development and qualitative-quantitative ratio aspect relative to the higher eukaryotes such as vertebrates. On the other hand the existence of two types of telomeric chromatin and the newly discovered Lia proteins associated chromatin makes it promising for new discoveries that would provide insight on the chromatin[1]. Tribolium castaneum or differently known as red flour beetle would be the second model organism I would choose to study the genetic expression and epigenetic mechanisms of the components of the chromatin such as histone and the DNA. The main reasons for this choice are its phenotypic plasticity, epigenetic effects controlling their gene expression such as temperature, diet etc, accessibility, low cost, the easy measurability of the trans-generational effects due to their morphogenetically different development stages, expression of the antimicrobial peptides due to the histone acetylation and are very practical for DNA methylation that directly affects the chromatin due to their high amount of euchromatin [2]. The last model organism I would use is Tetrodontidae or putterfish as a higher eukaryotic organism, sharing genome similarity with humans, having a pure DNA (low concentration of “junk DNA”) and exhibition of interesting chromatin phenomena such as chromatin elimination [3].

References

1.http://genesdev.cshlp.org/content/7/12b/2641.full.pdf retrieved from Google Resource on 06.03.2018

2.https://www.sciencedirect.com/science/article/pii/S0079610715000346 retrieved from Google Resource on 06.03.2018

3.https://www.ncbi.nlm.nih.gov/pmc/articles/PMC430906/