1. The use of 16S rRNA gene sequences to study phylogeny and taxonomy

2. The 16S rRNA gene is a section of prokaryotic DNA found in all bacteria and archaea. This gene codes for an rRNA, and this rRNA in turn makes up part of the ribosome. The first 'r' in rRNA stands for ribosomal. The ribosome is composed of two subunits, the large subunit (LSU) and the small subunit (SSU). While there are also associated proteins helping to make up the functional units of the ribosome, in general, in bacteria, the SSU is coded for by the the 16S rRNA gene, and the LSU is coded for by the 23S rRNA & 5S rRNA genes. What is the 16S rRNA Gene?

3. The use of 16S rRNA gene sequences to study bacterial phylogeny and taxonomy has been by far the most common housekeeping genetic marker used for a number of reasons. These reasons include : 1- its presence in almost all bacteria, often existing as a multigene family, or operons 2- the function of the 16S rRNA gene over time has not changed, suggesting that random sequence changes are a more accurate measure of time (evolution). 3- the 16S rRNA gene (1,500 bp) is large enough for informatics purposes The use of 16S rRNA gene sequences to study bacterial phylogeny and taxonomy

4. Dendrogram showing the genetic relationships of many of the major groups of clinically important organisms based on the 500-bp 16S rRNA gene sequence.

5. rRNA sequences play a central role in the study of microbial evolution and ecology. Particularly, the 16S rRNA genes have become the standard for the determination of 1- Phylogenetic relationships 2- The assessment of diversity in the environment 3- The detection and quantification of specific populations The rRNAs combine several properties which make them uniquely suited for such diverse applications.

6. First, they are universally distributed, allowing the comparison of phylogenetic relationships among all extant organisms and thus the construction of a “tree of life.” Second, the rRNAs are generally thought to be part of a core of informational genes which are only weakly affected by horizontal gene transfer (HGT), so their relationships provide a solid framework for the assessment of evolutionary changes in lineages. Third, the rRNAs are functionally highly constrained mosaics of sequence stretches ranging from conserved to more variable.

7. This enables the design of PCR primers and hybridization probes with various levels of taxonomic specificity and is exploited in microbial ecology when the number and distribution of different rRNA genes are taken as a measure of diversity.

8. How to design your PCR primer : You should put a criteria to selct the primers such as : The length of the primer, overall coverage of variable regions and amplicon length and Annealing temperatures

9. 1- Bacterial genomic DNA is extracted from whole cells by using a standard method or a commercial system (e.g., PrepMan DNA extraction reagent; ABI). The DNA is used as the template for PCR to amplify a segment of about 500 or 1,500 bp of the 16S rRNA gene sequence. Broad-based or universal primers complementary to conserved regions are used so that the region can be amplified from any bacteria. The PCR products are purified to remove excess primers and nucleotides; several good commercial kits are available (e.g., QiaQick PCR purification kit [Qiagen] and Microcon-100 Microconcentrator columns

10. 2- The next step is a process called cycle sequencing. It is similar to PCR in that it uses DNA (purified products of the first PCR cycle) as the template. Both the forward and reverse sequences are used as the template in separate reactions in which only the forward or reverse primer is used. Cycle sequencing also differs from PCR in that no new template is formed (the same template is reused for as many cycles as programmed, usually 25 cycles) and the product is a mixture of DNA of various lengths. This is achieved by adding specially labeled bases called dye terminators (along with unlabeled bases), which, when they are randomly incorporated in this second cycle, terminate the sequence. Thus, fragments of every size are generated. As each of the four added labeled terminator bases has different fluorescent dye, each of which absorbs at a different wavelength, the terminal base of each fragment can be determined by a fluorometer.

11. 3-The products are purified to remove unincorporated dye terminators, and the length of each is determined using capillary electrophoresis (e.g., ABI PRISM 3100 genetic analyzer with 16 capillaries or ABI PRISM 310 genetic analyzer with 1 capillary) or gel electrophoresis (e.g., the Visible Genetics system). Since we then know the length and terminal base of each fragment, the sequence of the bases can be determined. The two strands of the DNA are sequenced separately, generating both forward and reverse (complementary) sequences. An electropherogram, a tracing of the detection of the separated fragments as they elute from the column (or are separated in the gel) in which each base is represented by a different color, can be manually or automatically edited. It is possible to have the fragments of various lengths so well separated that every base of a 500-bp sequence can be determined. When ambiguities occur, most of them can be resolved by visual reediting of the electropherogram.

12. 4- The generated DNA sequences are usually assembled by aligning the forward and reverse sequences. This consensus sequence is then compared with a database library by using analysis software. Some systems allow comparisons of the single forward or reverse sequences. Well-known databases of 16S rRNA gene sequences that can be consulted via the World Wide Web are GenBank (http://www.ncbi.nlm.nih.gov/), the Ribosomal Database Project (RDP-II) (http://rdp.cme.msu.edu/html/), the Ribosomal Database Project European Molecular Biology Laboratory (http://www.ebi.ac.uk/embl/), Smart Gene IDNS (http://www.smartgene.ch), and Ribosomal Differentiation of Medical Microorganisms (RIDOM) (http://www.ridom.com/).http://www.ncbi.nlm.nih.gov/http://rdp.cme.msu.edu/html/http://www.ebi.ac.uk/embl/http://www.smartgene.chhttp://www.ridom.com/

13. The decontamination procedure and the broad-range real-time PCR method allow rapid detection, quantification, and classification of several clinically important bacteria and may facilitate rapid detection of local and systemic infection. Using real time PCR to analyse rRNA

14. Bacterial genomic DNA was isolated by use of the MasterPure DNA purification reagent set (Epicentre Technologies). Briefly, pellets from bacterial cultures were resuspended in 300 μL of Tissue and Cell Lysis Solution containing proteinase K (160 mg/L) and incubated at 65 °C for 15 min. After the lysis process, RNase A (160 mg/L) was added for 1 h at 37 °C. We then added 150 μL of MPC Protein Precipitation Reagent and centrifuged the mixture at 20 000g for 10 min. The supernatant was transferred to a clean microcentrifuge tube, and the DNA was precipitated with isopropanol. After two washes with 750 mL/L ethanol, the DNA pellet was resuspended in water for real-time PCR analysis.

15. The broad-range real-time PCR was first set up to amplify the Escherichia coli 16S rRNA gene fragment with the universal primers p1370/p201 (13). The template DNA was added into the reaction mixture containing 25 μL of 2× SYBR Green PCR master mixture (Applied Biosystems), 1 μL of p201 (5 × 10 −6 M; 5′- GAGGAAGGIGIGGAIGACGT-3′), and 1 μL of p1370 (5 × 10 −6 M; 5′- AGICCCGIGAACGTATTCAC-3′) in a final volume of 50 μL. PCR was performed with the GeneAmp 5700 Sequence Detection System (Applied Biosystems). After initial activation of AmpliTaq Gold DNA polymerase at 95 °C for 10 min, 40 PCR cycles of 95 °C for 15 s and 60 °C for 1 min were performed. Immediately after PCR amplification, we performed melting curve analysis (16) by cooling the reaction to 60 °C and then heating it to 95 °C in an ∼ 20- min period. The SYBR Green I fluorescence (F) was measured continuously during the heating period, and the signal was plotted against temperature (T) to produce a melting curve for each sample. The melting peak was then generated by plotting the negative derivative of the fluorescence with respect to temperature against temperature (−dF/dT vs T). The melting temperature (T m ) was considered the peak data point in the melting curve analysis.1316

17. http://www.ncbi.nlm.nih.gov/pmc/articles /PMC93223/