Transcript PCR analysis

The use of 16S rRNA gene sequences to
study phylogeny and taxonomy
What is the 16S rRNA Gene?
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.
The use of 16S rRNA gene sequences to study bacterial
phylogeny and taxonomy
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
Dendrogram showing the genetic
relationships of many of the major
groups of clinically important
organisms based on the 500-bp 16S
rRNA gene sequence.
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.
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.
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.
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
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
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.
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.
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/).
Using real time PCR to analyse rRNA
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.
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.
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 × 10−6M; 5′GAGGAAGGIGIGGAIGACGT-3′), and 1 μL
of p1370 (5 × 10−6M; 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 ∼20min 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
http://www.ncbi.nlm.nih.gov/pmc/articles
/PMC93223/