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PCR.ppt
1. Polymerase Chain Reaction
PCR is an in vitro technique for the amplification of a
region of DNA which lies between two regions of known
sequence.
2. What you need for PCR (inhouse/commercial are same
components)
1. Buffer
2. Mg++ ions
3. dNTPs (Nucleotides)
4. Enzyme
5. Primers
6. DNA/RNA template
7. Water
8. Consumables and PCR Machine
(Conventional/Real-Time)
3. 1. Buffer
Buffer
Stabilizes the DNA polymerase, DNA template,
and nucleotides
500 mM KCl
100 mM Tris-HCl, pH 8.3
Triton X-100 or Tween
Commercially available/can be prepared in lab
4. 2. Mg++ ions
Bivalent cations, magnesium or manganese ions;
generally Mg2+ is used, but Mn2+ can be used for
PCR-mediated DNA mutagenesis, as higher Mn2+
concentration increases the error rate during DNA
synthesis
Monovalent cation potassium ions
Commercially available/can be prepared in lab
5. 3. dNTPs (Nucleotides)
Deoxynucleoside triphosphates (dNTPs, sometimes
called "deoxynucleotide triphosphates"; nucleotides
containing triphosphate groups), the building-blocks
from which the DNA polymerase synthesizes a new
DNA strand.
Commercially available/can be prepared in lab
6. 4. Enzyme
Taq polymerase, a DNA polymerase that is heat
resistant, so that it can remain intact during the
DNA denaturation process.
Commercially available/can be prepared in lab
7. 5. Primers
• PCR amplification is achieved by using oligonucleotide primers.
– These are typically short, single stranded oligonucleotides which are
complementary to the outer regions of known sequence.
• The oligonucleotides serve as primers for DNA polymerase and the
denatured strands of the large DNA fragment serves as the template.
– This results in the synthesis of new DNA strands which are
complementary to the parent template strands.
– These new strands have defined 5' ends (the 5' ends of the
oligonucleotide primers), whereas the 3' ends are potentially
ambiguous in length.
9. 1. Conventional PCR
The final result of the traditional PCR
procedure is a gel with a series of bands:
Bands can be compared against each
other, and to known size-standards, to
determine the presence or absence of a
specific amplification product.
10.
11. In-house PCR Assay
• Literature review
• Analysis of primers/probes
• Nucleic Acid Extraction
• PCR with Control samples
• Standardization
• Measuring of Sensitivity and Specificity
• Efficiency
• Repeatability
• Comparison with commercial kits
• Inter Lab Comparison of Results
12. Typhoid In-house PCR
• Literature Review
Method
Test
(culture site)
Sensitivity Specificity Comments
Microbiologic
Blood 30-70% 100%
A number of advantages
Sensitivity increases with volume
Bone marrow 80-95% 100% Considered gold standard but impractical
Stool 30-50% - -
Urine 15-50% - -
Genomic PCR 50-95% variable Issues of DNA isolation
Immuno Assay
Widal (Ab) 70-100% 30-85% Specificity decreases in endemic zones
IDL Tubex®
(Anti-LPS IgM)
60-85% 55-80%
Many small studies in general less
sensitive but more specific than Widal
Typhidot®
(Anti-OMP
IgG/IgM)
75-90% 50-90%
TPTest
IgA Assay
>90% >90%
Sensitive, specific, minimal laboratory
requirements
23. Literature Review Findings
• Salmonella typhi
• Salmonella Paratyphi A
• Selection of conserved genes for primers
• Primers for Flagellin Gene and other
genes
24. • DNA isolation
• Different techniques used
• Kit based DNA isolation
• In-House DNA isolation
Literature Review Findings
25. Procurement of DNA isolation Reagents and all
reported Primers in literature
26. Experimental Work
• Collection of samples
• Testing of different DNA isolation methods
reported in literature for DNA isolation
• Testing of different primers methods
reported in literature for PCR
28. Conclusion
• Selection of best DNA isolation method
(commercial/in-house)
• Selection of best PCR primers after
experimental findings
• In-House PCR Assay ready
