16S Ribosomal RNA Sequencing and Phylogenetic Tree Construction

The purpose of this experiment is to form phylogenetic tree between five different kinds of prokaryotic organisms by using 16S rRNA analysis.
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.
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).

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.
[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
[2]  Woese CR, Fox GE (November 1977). “Phylogenetic structure of the prokaryotic domain: the primary kingdoms”. Proceedings of the National Academy of Sciences of the United States of America. 74 (11): 5088–90. Received on May 14, 2017
[3] Case RJ, Boucher Y, Dahllöf I, Holmström C, Doolittle WF, Kjelleberg S (January 2007). “Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies”. Applied and Environmental Microbiology. 73 (1): 278–88. Received on May 14, 2017
[4] Schmidt TM, Relman DA (1994). “Phylogenetic identification of uncultured pathogens using ribosomal RNA sequences”. Methods in Enzymology. Methods in Enzymology. 235: 205–22. Received on May 14, 2017
[5] Hodge T, Cope M (2000). “A myosin family tree”. Journal of Cell Science. 113 (19): 3353–4. Received on May 14, 2017
[6] (http://www.ebi.ac.uk/Tools/msa/tcoffee/).

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