Computational Biology Exam Sample

1) What is the difference between application and program? Explain in one (1) sentence max. (5
pts)
2) Why would we prefer command line based tool over graphical user interface (GUI) based tool?
Explain in two (2) sentences max. (10 pts)
3) Why is the Basic Local Alignment Search Tool called a heuristic algorithm? Explain in one (1)
sentence max. (5 pts)
4) What is the difference between database search and de novo search in mass spectrometry based
search tools ? Explain in one (1) sentence max. (5 pts)

Exam Questions about Computational Biology

1) GFF (General feature format) files are widely used in data annotation, describing genes and
other molecules. Find the example GFF formatted file in your cms (Q1.gff).
a) Search and print every line that contains the word “overlap” using find command in cmd
(10 pts)
b) Redirecting (pipeline) the output of the problem (a) to the input of a new find command,
find all the lines that contain the word “chrXII” (10 pts)
c) Redirect the output of problem (b) to a new file named “Answer1.txt” (5 pts)
d) Put all the command lines that you used in this question to a file named
“Answer1_cmdline.txt”. Send both of the files.
2) Find the “blast_exam” folder in your cms.
a) Index the database file (7 pts)
b) Blast the query to database using “blastp” but use a switch to filter out all the alignments
that has identity below “95.3” percent (the number “95.3” is a float value) (13 pts)
(Hint: Help is always given on command line prompt to those who ask for it)
c) Put all the command lines that you used in this question to a file named
“Answer2_cmdline.txt”. Send this file and screenshots.
3) Find the “xtandem.zip” file in your cms. Inside the “src” folder, you have “exam_spectra.mgf”
and “exam_protein.fasta” files. Don’t move any file.
a) Index the database file (from “src” folder). (5 pts)
b) Fix “taxonomy.xml” and “input.xml” files in such a way that you can search
“exam_spectra.mgf” in “exam_protein.fasta” (mgf and indexed fasta files will be used
from src folder) (18 pts)
c) Set “fragment monoisotopic mass error” option to 0.7 and run xtandem program (7 pts)
d) Put all the command lines that you used in this question to a file named
“Answer3_cmdline.txt”. Send “Answer3_cmdline.txt”, “default_input.xml”,
“taxonomy.xml”, “input.xml” and screenshot files.
Please put all your files and answers in separate folders (e.g. folder “a1”, “a2”…)
then zip them into one file with the name “yourname_yourstudentid”.zip.

ComputationalComputational Biology Exam Folder

Reliability of Renewable Energy

Renewable energy or as they are differently known the alternative energy sources has become one of the most debatable topics of our times related to their reliability. Hydrogen fuel, biomass, hydropower, solar power, geothermic energy and nuclear power are some of the most common types of renewable energy. On the other hand the fossil fuels (a not renewable energy type) still continue to dominate the energy sector, apart from the fact that it is a troublesome type of energy and nonsustainable. Furthermore the fossil fuels have a hazardous effect toward the environment and still overshadow the alternative energy sources about their reliability.
Breeder reactor is the best epitome of the type of the renewable energy that arises most of the reliability questions. A breeder reactor is a device which uses the principle of heat assisted atom splitting in order to generate power. Is is self sufficient and it consists of a coolant that serves as a core protecter from overheating. They are able to create 30% more fuel than the amount they consume. In addition, those nuclear reactors need only an infrequent uranium addition about energy production. The uranium, which is the first material about conversion is a sustainable material that is ubiquitously found on the seas and even minute amounts of uranium can generate ten times more energy than a ton of coal. One of the most crucial advantages that the breeder reactors garantuee is the ability of energy conversion from the uranium waste and the depleted nuclear weapons’s uranium. The breeder reactors garantuee a more inexpensive method compared to the more reliable methods such as fuel, because of the reusability of the uranium as first work material.
One of the most critical problems that arises the problem with the reliability of this renewable energy form is the accident possibility that is posed because of the high temperature and the fast pace on the work paradigm. Furthermore, the ability of plutonium that has a half life of 24000 years, to persist for a long time poses a threat for cancer in case of inhalation. Also the byproducts of the breeder reactors tend to be dangerous for human health. On the other hand, the newest nuclear technologies have developed plans to overcome the challenges posed from these breeders.

References:

http://www.altenergy.org/renewables/renewables.html

Master Application Reference Models

To whom it may concern,

It is my pleasure to support Student X who is applying for a scholarship program by writing this recommendation letter.

I have known Student X during her volunteering work in my hospital during the periods: June-September 2014 and June-September 2015 and I can assure that she is a very hardworking girl.

She has shown to have a dedicated and disciplined personality. Her passion for Molecular Biology and her tenacity to achieve highest goals during intern program impressed me and other laboratory members. During her work in the laboratory, Student X demonstrated a combination of her deep interest and curiosity for research techniques with relevant knowledge and analytical skills. She was also very curious about developing more knowledge in clinical aspect of the eye illnesses. Also, she integrated well in team work by taking responsibilities and helping her teammates.

I hold Student X in top 5% out of hundreds intern students we have had in our hospital that collaborates with a Molecular Biology and Genetics Lab in Athens, Greece . I think that she has successfully built a rich and complete background of a successful student. I believe that during these years she has planted strong concepts of Genetics, Cell Biology and Cloning Techniques.

I strongly recommend her for this scholarship program. I believe that with her ambition, passion for science and correctness, she will succeed . Please do not hesitate to contact me for any further question about my student Student X.

Sincerely,

Prof. Dr ……

General Director of ……,

Telephone Number

E mail adress

Adress

Reference Samples for Science Students

To whom it may concern,

I am writing this recommendation letter to support the application of Student X who was my research student during the period June-September 2016 and June-September 2017.

Since the beginning I noticed that she was very careful in listening and didn’t hesitate to ask in order to understand in depth the new information received. In regard to her laboratory performances, she was one of the most prepared intern students. Not only that, but I realized in our open discussions that she was learning extra information even independently. During this time, she showed curiosity about some specific topics. One of them was structural and functional adaptation in different conditions. She presented a high interest in how the human beings change their expressed structural characteristics in order to adapt to a new specific condition. She finalized this by presenting a topic at the end of the program named: “Environment shaping us” where she presented how we should understand the adaptation process, and some of the molecular mechanisms how it occurs. Other fields of interest were mechanism of native immunity and the use of stem cells nowadays. Another point that I would like to emphasize at my student, is her desire and passion of working in the laboratory. In our lab, she learned how to prepare micro and macro samples and was trained in how to make the identification of certain tissues under the microscope, performed ELISA test, histochemical tests and Western Blot. She was always asking me extra hours to improve her “microscope” skills showing an outstanding passion for laboratory work. Knowing her ambition of continuing a career in cancer research, I think that Japan would be like a trampoline for her future ambitions in life. Her academic background, together with curiosity, hard work and willpower which characterize her nature are the best indicators of her success . Please feel free to contact me if you have any question about my intern student Student X.

Name of the Reference provider

Mail adress

Adress

Reference Samples for Master Application of Molecular Biology and Genetics Students

To whom it may concern,

Please accept the following as a committee letter in support of the the student Student Y , student of Molecular Biology and Genetics in University Z.

I have become well acquainted with Student Y during the past 4 years she studied Bachelor and she has done quite well academically. Apart from the fact that during a part of the study her record contains some mediocre grades, Student Y has done an amazing work on the research laboratory dedicating most of the hours of her day to the experiments involved in my lab whose major interest is the study of the cancer mechanism and the stem cells involvement in this process. During those 4 years, she has displayed maturity, great leadership skills in the laboratory and great knowledge acquired from the self study. Furthermore her presence on my laboratory was both inquisitive and energetic and she is a good team player. Her major success indicator is the fact she used to work to support her studies and managed on the same time to work voluntarily in my research laboratory to manipulate the mesenchymal cells and also attend her lectures.

She is an extraordinary young woman who has the intellectual horsepower to do amazing research and has the personal skills that make her a great person, therefore she has my highest recommendation. If you have any further questions, please do not hesitate to contact me.

Professor Professor X

Mail adress

University Z,

City, country

Differences between Prokaryotes and Eukaryotes

Both prokaryotes and eukaryotic genomes are DNA genomes. Prokaryotic genome is usually found on the cytoplasm and consists of only one chromosome, while the eukaryotic one consists of multiple chromosomes and most of the eukaryotic genome is found on the nucleus. Prokaryotic genome is less complex compared to the eukaryotic one which generally consists of longer genes. The coding sequences compart nearly the 90% of the prokaryotic genome and only the 3% of the eukaryotic genome. The prokaryotic genome tends to be circular, while the eukaryotic genomes are generally linear. The prokaryotic genomes are always haploid, but the eukaryotes can be either diploid or haploid. Compared to the eukaryotic genomes, the prokaryotic genomes have a high rate of protein coding genes. Furthermore, the eukaryotic genome has hundreds of rRNA genes compared to the prokaryotic ones that have 1-10 rRNA genes. Eukaryotic genomes have a higher rate of repeated sequences, end telomeres and introns compared to the prokaryotic genomes that rarely consist of those parts. Also origin of replications usually are numerous on eukaryotes but found only once in prokaryotes.[1]. Both eukaryotic and prokaryotic gene structures include similar sequence elements necessary for gene expression. They both involve regulatory elements such as promoters, silenceres and enhancers usually on their ends and contain ORI. Also the reading strands of both eukaryotic and prokaryotic strands is on the 5′-3′ direction. Eukaryotic genes differ from prokaryotic genes because introns are absent in prokaryotic genes and in prokaryotic genes there is no such a phenomenon such as alternative splicing. Also eukaryotic genes consist of more regulatory regions compared to the prokaryotic ones. In prokaryotes the genes can sometimes organize themselves in a polycistronic operon. Riboswitches also are some regulatory regions of the prokaryotic genes that lack on the eukaryotic genes. The main difference of the RNA polymerase II assisted transcription initiation is that in prokaryotes only DNA containing promoter and polymerase II are needed, while in eukaryotes several transcription initiation factors and proteins more are needed, such as TF, TBP, TFIID, TAF[2].

References

1-http://www.differencebetween.com/difference-between-prokaryotic-and-vs-eukaryotic-genome/

2 – https://www.ncbi.nlm.nih.gov/books/NBK9935/

Fosforilimi Oksidativ dhe Sinteza e ATP-s 

Fosforilimi oksidativ dhe fotofosforilimi sigurojnë me te shumtën e ATP-së në shumicën e gjallesave. Fosforilimi oksidativ është termi përdorur për dukurinë e reduktimit te oksigjenit në nje molekulë uji ku elektronet dhurohen nga NADH  dhe FADH2. Fosforilimi dhe fotofosforilimi janë të ngjashme nga ana mekanike për tre arsye: 

1-rrjedha e elektroneve në kanale të suportuar nga membrana 

2-potenciali elektrokimik midis membranave 

3-sinteza e ATP-së me anë të protonëve që ndodhen në gradientin e kundërt 

Pjesët përbërëse të mitokondrisë 

1-Membrana e jashtme-e përshkrueshme nga molekulat e vogla dhe jonet 

2-Membrana e brendshme-e pakalueshme nga jonet ose molekulet e vogla duke përfshirë dhe jonin hidrid. Ajo përmban transportuese elektronë që janë përgjegjës për procesin e respirimit, transportuesë të tjerë, sintazen ATP, translokatorin ADP-ATP 

3-Matriksi-Përmban kompleksin e piruvatit dehidrogjenues, enzimat për ciklin e acidit citrik, enzimat për beta-oksidimin e acideve të yndyrshëm, ATP, ADP, jonet e kalciumit, kaliumit. 

Fosforilimi oksidativ fillon me hyrjen e elektronëve në zinxhirin respirativ. Enzimat dehidrogjenues mbledhin elektronë nga ciklet katabolike dhe i udhëzojnë ato drejt NAD, NADP ose FAD që njihen ndryshe si pranueset universale të elektronëve. Enzima dehidrogjenuese e lidhur me NAD shërben për të hequr dy atome hidrogjeni prej substrateve të tyre. Njëri nga këto hidrogjene trasmetohet drejt NAd+ kurse tjetri ngelet në gjendje të lirë si jon hidrid. NADH mbledh elektrone nga ciklet katabolite kurse, NADPH mundëson reaksionet anabolike me elektrone.  

 Flavoproteinat pë rmbajnë një nukleotidë flavine (FMN ose FAD). Nje nukleotidë flavine e oksiduar pranon një  elektron dhe në kë të më nyrë prodhon FADH+ ose FMNH+ ose mund të pranojë dy elektrone pë r të prodhuar FADH2 ose FMNH2. Potenciali i reduktimit ë sht i varur ndaj lidhjes me proteinat e ndë rlidhura me të .  

Kalimi i elektronave në pë r transmetuesit që pë rmbajnë membranë .  

Transferimi i elektronë ve në  fosforilimin oksidativ mund të ndodhë në pë rmjet transfertë s direkte të elektronë ve, transfertë s si atom hidrogjeni ose si jon hidrid.  

Molekulat që transferojnë elektrone: 

NAD 

Flavoproteinat 

Jubikuinonet 

Citokroma 

Proteinat që  pë rmbajnë hekur dhe sulfur 

 

Jubikuionet 

Janë  koenzima të  lidhura me yndyra të tretshme Q benzokinone dhe pë rmbajnë   një zinxhir anë sor isoprenoid. Ato pranojnë 1 ose 2 elektrone. Mund të shpë rndajë edhe elektrone edhe protone që kanë një rol të rë ndë sishë m në pompë n protonike.  

Citokroma 

Ë shtë e aftë pë r të kapur dritë n e diellit në pë rmjet grupit heme që pë rmban, si a, b apo c. Kofaktorë t a apo b lidhen në një më nyrë kompakte me proteinat, kurse grupet heme të llojit c lidhen në më nyrë  kovalente në pë rmjet lidhjeve Cys. Citokromat e llojit a ose b janë poteina integrale kurse ato të llojit c mund të kalojnë lehtë sisht pasi janë të tretshme.  

Zinxhiri respirativ i mitokondrisë  

Kahu i procesit synon drejt rritjes së potencialit reduktues.  

NADH-Q-citokroma b-citokroma c1-citokroma c-citokroma a-citokroma a3-oksigjeni 

 Komplekset multienzimatikë  që luajnë rol në  transfertë n e elektronë ve 

Kompleksi I: NADH-Jubikuinone 

Deri në 42 zinxhirë anë sorë , duke pë rfshirë  flavoproteina si dhe të paktë n 6 qendra me hekur dhe sulfur.  

 

IMViC Tests and Catalase Test

Aim:
The aim of this experiment is to determine kind of Enterobacteriaceae bacteria that are found in the tubes labelled as α and β by using IMViC tests.
Introduction:
In microbiology, kind of bacteria in Enterobacteriaceae of determination is done by using some methods. The rapid and useful test is IMViC. For checking danger or safety of water or food, this test is so important. (1)
Enterobacteriaceae is a huge family in bacteria. They have Gram-Negative bacteria including harmless and pathogenic kinds. Coliforms are found in this family. Coliforms are separated according to fermenting lactose with gases and acid. Coliforms are rod-shaped, Gram-Negative, non-spore forming bacteria. They mostly live in aquatic environment and fecal contamination is considered with coliforms. Because of that, they are so important as indicator for these purpose. They are used for pollutions or determining pathogenic organisms. For example, Escherichia coli is one of the most known coliform like Klebsiella pneumonia and Enterobacter aerogenes. Coliforms are obtained by using IMViC test. IMViC include some serial test to determine species of coliform. Each letter except “i” refers to first letter of tests. (1, 2 and 3)
Indole test is first test and used for obtaining present of indole amino acid. Some coliforms use tryptophan as substrate and they produce indole after reaction. Kovac’s reagent, so dangerous with air, is used for that. The reagent reacts with presence of indole and gives pink/red colour on top layer of the tube. E.coli is one of bacteria giving positive result for indole test. Many Bacillus gives negative result like this experiment. (4, 5 and 6)
Methyl-red or shortly MR test is based on pH value in solution. It acts like an indicator giving red colour with acidic solution. Glycoses is first step for generating energy for living organisms. At end of glycoses, pyruvates or pyruvic acid are produced from glucose. Naturally pyruvic acid makes acidic. Bacteria convert them to more stable and safer acidic forms such as formic acid, acetic acid or lactic acid. Eventually acidic media causes change of colour with reagent. E.coli forms acidic media and it causes positive or red colour at MR test. B.subtilis demonstrates negative result or yellow-brown colour. (4, 5 and 6)
VP test or Voger-Proskauer test is the other step of IMViC. VP is for determined existence of acetone in bacterial culture. Some bacteria live by using glucose and turning into acetylmethylcarbinol, shortly acetone. Two solutions are added for that. First one is alpha-naphthol. It binds to acetone and the second solution, potassium hydroxide forms cherry red colour if it is positive. Negative is to see yellow-brown colour for this test. B.subtilis is in positive group; however same test gives negative with E.coli. (4, 5 and 6)
Citrate Utilization test is a test to control bacteria use whether citrate or not as energy-carbon source. When bacteria are added into a media that have sodium citrate and pH indicator like bromothymol blue as basic test materials, citrase enzyme in bacteria breaks down citrate to oxaloacetate and acetate. Oxaloacetate becomes to pyruvate and CO2. On the other hand, after some reaction with acetate, sodium citrate and ammonium salts cause changes in alkaline pH value. These changes forms colour media to blue from green if it is positive like in B.subtilis. Negative result doesn’t demonstrate any growth on media. E.coli can be given an example for negative. (4, 5 and 6)
Catalyst test is one of simple tests. Some bacteria can break down H2O2 to water for producing energy. In this test, H2O2 is dropped onto cells directly and if bubble is observed, it means test is positive. For E.coli, it is variable, but for B.subtilis, it is positive. (4, 5 and 6)
Material and Methods:
Indole Production Test,
0.3 ml of Kovac’s reagent was taken and transmitted into the sample tubes α and β with Tryptone in hood. After a while, colours of samples were observed.
Methyl-Red Test,
1 ml of methyl-red indicator was added into the samples with glucose phosphate broth and the samples were waited for colour change.
Voges-Proskauer Test,
0.9 ml of solution A and 0.3 ml of solution B of Barrit’s reagent were added into samples and they were observed.
Citrate Utilization Test,
Tubes with Simmon’s Citrate Agar were transmitted into test samples and they were waited for 48 hours at 37°C in incubator. After that, they could be observed.
Catalase Test,
3% H2O2 solution was dropped onto bacterial colonies of the test samples that were incubated at small amounts onto a plate before. The samples form bubble was observed.
Results:
Tests
Samples Indole Methyl-red Voger-Proskauer Citrate Utilization
α – + – +
β + – – –
Table 1- the results of IMViC tests for 2 strains (α and β)
Catalase Test
α +
β +
Table 2- Catalase Test for 2 strains (α and β)
α and β refer to E.coli and B.subtilis as unknown samples.
Discussion:
In this experiment, it is aimed to determine kinds of the samples in coliforms by using some serial tests according to metabolic activities.
According to the results of this experiment, it can be seen that α and β have different metabolic activity. For first test for IMViC, indole test was done for each sample. Indole test is bacteria use tryptophan and turns it into indole amino acid. At presence of indole in media, Kovac’s reagent gives a pink layer on top of tube by forming a complex with indole. The sample β gave positive for indole test and the bacteria in sample β uses tryptophan for its metabolism. E.coli is one of kinds of bacteria that are positive at indole test. (5,6)
At second test, methyl-red test, reagent is used for obtaining pH value in sample basically. Methyl red acts as indicator to shows colour change according to pH value. Some microorganism metabolizes glucose to pyruvic acid and it makes environment acidic, but they convert pyruvic acid to some forms such as lactic acid, acetic acid or formic acid for preventing accumulation of pyruvic acid. However environment is still acidic and methyl red can give a reaction. Positive test that bacteria produce low pH value about 4 forms red colour from yellow. Negative gives yellow with more basic environment. α and β indicate different and unexpected result for MR test. The sample α may be B.subtilis and the sample β may be E.coli by comparing with the other tests, however α and β show unexpected results. There may be experimental error. The tubes that have been labelled as α and β could have been confused before experimental process. α and β may have been confused again at observation part. These kind of experimental errors may be and also contaminated culture with the other bacterial cultures or wrong aseptic techniques may give these results. (5, 6)
VP test or Voger-Proskauer is used for identified presence of acetone in bacterial culture. At VP is applied with two solutions for reacting with acetone and giving colour. These are alpha-naphthol and potassium hydroxide. If bacteria turns glucose into acetylmethylcarbinol at digestion, alpha-naphthol reacts and potassium hydroxide gives cherry red colour for positive result. At negative result, yellow-brown or copper colour indicates. In this experiment, there is no positive result. Both cell cultures don’t give acetone after their metabolic activity as expected. E.coli and B.subtilis must have negative result after VP test. (5, 6)
Citrate utilization test is to detect use of citrate by bacteria. Some bacteria use citrate for their metabolic activity. Citrate utilization test uses reagent that can change their colour after reaction in pH value. If test is positive, a change in colour forms from green to blue. B.subtilis can cause this kind of change on media. The sample α can be seen a B.subtilis culture. E.coli culture doesn’t look like that like at β culture. (5, 6)
Catalyse test is the easiest test than the other tests. Drops of H2O2 were poured on two cultures that were found on different places of same slide. Bubbles were observed at both samples. Already forming bubbles were expected at B.subtilis. The sample α that is thought it is B.subtilis shows same results with catalyse test. On the other hand, E.coli bacteria were variable about forming bubbles. In this experiment, β sample is E.coli that can form bubble with H2O2. (5, 6)
Except MR results, samples can be identified which kind of bacteria exist in tubes. According to Table 1 and Table 2, α sample is B.subtilis and β sample is E.coli. (5,6)

Reference:
[1] Received on 24 May,
MacFaddin J.F. 2000. Biochemical Tests for the Identification of Medical Bacteria, 3rd ed. Lippincott Williams & Wilkins, Philadelphia, PA, USA.
[2] Received on 24 May,
Don J. Brenner; Noel R. Krieg; James T. Staley (July 26, 2005) [1984 (Williams & Wilkins)]. George M. Garrity, ed. The Gammaproteobacteria. Bergey’s Manual of Systematic Bacteriology.  2nd ed. New York: Springer.
[3] Received on 24 May,
(http://www.moldbacteriafacts.com/what-are-bacteria/what-is-coliform/)
[4] Received on 24 May,
MacFaddin, Jean F. “Biochemical Tests for Identification of Medical Bacteria.” Williams & Wilkins, 1980, pp 173 – 183.
[5] Received on 24 May,
(http://www.microbiologyinfo.com/biochemical-test-and-identification-of-e-coli/)
[6] Received on 24 May,
(http://www.microbiologyinfo.com/biochemical-test-and-identification-of-bacillus-subtilis/)

 16S Ribosomal RNA Sequencing and Phylogenetic Tree Construction

Aim:
The purpose of this experiment is to form phylogenetic tree between five different kinds of prokaryotic organisms by using 16S rRNA analysis.
Hypothesis:
For forming phylogenetic tree, genomic DNA of each organism should be isolated and then interested regions of DNA (rRNA sequences or ribosomal RNA sequences) are amplified by PCR. For knowing these sequences, amplified DNA fragments are sequenced and phylogenetic tree is formed by using bioinformatics tools.
Introduction:
rRNA or ribosomal RNA is essential material that joins protein production. rRNA is produced from rRNA sequences or rDNA sequences. The importance of these rRNA sequences is to have very conserved regions and also differences that species have. These sequences are used for determined place of specie in taxonomy. (1)
There are three types of rRNA in prokaryotes. These are 5S, 16S and 23S. Without eukaryotes, 16S is often used for determination of phylogenetic tree or taxonomic places of organisms. For eukaryotes, 18S is used for that. rRNA sequences include some commonplace regions. As universal, some regions are same for all organisms. These regions are much conserved because of mutual and essential sequences. These conserved regions are named as highly conserved regions. The regions 16S includes differences is called hypervariable regions or hot-spots. These differences are used for taxonomy. (1, 2)
Size of rRNA genes is suit for bioinformatics applications and tools. Also conserved regions are universal and it helps to use universal primer for amplification of the gene. After years, researchers have a huge database about rRNA genes and it makes researches easy for them. Hypervariable and conserved regions can help by different ways for constructing phylogenetic tree. (2, 3)
Before construction of phylogenetic tree, interested rRNA genes are amplified by PCR for forming better results and working easier. Ribosomal RNA sequences are used as template DNA. Universal primers also make process easier by highly conserved regions. (4)
Phylogenetic tree is a figure that is used for showing evolutionary relativeness between living organisms. Similarity of genes is related with evolutionary origins. Figures on phylogenetic tree represent different relationships or closeness. Each node is a connection between braches and it demonstrates an ancestor. Branches are formed with differences among kinds. How far two kinds are on tree can be understood by shared nodes. In a tree, if there is a common ancestor for all organisms, this tree is named rooted tree or if there is no, it is named unrooted tree. (5)
Materials and Method:
Firstly, 16S rRNA sequence of DNA that was isolated before experiment was amplified by PCR process. After PCR components were calculated, components were mixed in an Eppendorf except enzyme. Enzymes were added into mixture and then lastly sufficient amount of water was added for completing mixture 50 µl. Finally mixture was placed into PCR machine.
Required Material Stock concentration Required concentration Volume (µl)
DNA 9.4 ng 250 ng 26.6
Primer Forward 10 µM 1 µM 5
Primer Reverse 10 µM 1 µM 5
dNTP (mix) 10 µM 200 mM 1
MgCl2 25 mM 2 mM 4
Taq Pol. 5 unit/µl 2.5 units 0.5
Buffer 10X 1X 5
H20 – – 2.9
Total: 50 µl

A PCR thermal cycle:
Denaturation (30 seconds at 950C)
Annealing (60 seconds at 680C)
Extension (5minutes at 720C)
Thermal cycle of PCR was repeated for 30 times.
Products of PCR were sequenced for rRNA analysis. Sequences were used for constructing a phylogenetic tree with help of T-Coffee (6).

Discussion:
This experiment was intended to make phylogenetic tree between five different organisms according to 16S rRNA sequences of them by firstly using PCR with universal primers then sequence and bioinformatics tools (6).
After constructing phylogenetic tree between five organisms, relativeness of organisms can be seen. Any common ancestor of five organisms cannot be observed on tree. This demonstrates that this phylogenetic tree is unrooted.
The nodes that have two branches from a branch on tree utilize an ancestor kind in mutual. As seen in the Figure 1, the closeness of Acinetobacter haemolyticus and Escherichia coli creatures as affinity is the closest organisms than the other organisms. The node on the right between these organisms releases that they have a common ancestor in the past. Enterococcus faecium is also close to these kinds partially. These three species seem to originate from a common ancestor. The node on the left provides this information to us about the other two organisms too. If Bacillus subtilis and Staphylococcus aureus are observed, these two kinds don’t have a close origin to each other can be seen. Also these two kinds also aren’t close to the other three kinds.
References:
[1] Smit S, Widmann J, Knight R (2007). “Evolutionary rates vary among rRNA structural elements”. Nucleic Acids Res. 35 (10): 3339–54. Received on May 14, 2017
(https://academic.oup.com/nar/article-lookup/doi/10.1093/nar/gkm101)
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(http://www.pnas.org/content/74/11/5088)
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(http://aem.asm.org/content/73/1/278)
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(http://www.sciencedirect.com/science/article/pii/0076687994351422?via%3Dihub)
[5] Hodge T, Cope M (2000). “A myosin family tree”. Journal of Cell Science. 113 (19): 3353–4. Received on May 14, 2017
(http://jcs.biologists.org/content/113/19/3353.full)
[6] (http://www.ebi.ac.uk/Tools/msa/tcoffee/).