Download PCR

Polymerase Chain Reaction
(PCR)
Nahla Bakhamis
Multiple copies of specific DNA sequences;
‘Molecular Photocopying’
Polymerase Chain Reaction
• 1983;
In vitro enzymatic amplification of specific DNA sequences from the
genome (2 regions of known sequence).
As many as billion times
C T T A C C G T G G T A A A T C G
G A A T G G C A C C A T T T A G C
PCR Properties:
Rapid & easy.
Sensitive.
Robust.
Widespread applications;
Bioinformatics, forensics, medicine and genetic research.
PCR uses:
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Replacement of cloning.
Diagnosis of chromosomes abnormalities (QF-PCR).
Diagnosis of single gene defect.
Searching for genes and mutations.
Cancer genetics.
PCR requirements:
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Known DNA seq of target region.
Primers 18-25 bases.
Thermo-stable DNA polymerase Taq-polymerase
dNTPs
Thermal cycler.
Reagents for PCR reaction:
• DNA template (1-5µL).
• 2 primers (1-3µL); if excess Primer-dimer
- complementary to 3’ end
- length 18-25 bp
- CG content 45-60%
Master mix:
a. Taq-polymerase
b. dNTPs – deoxynucleoside triphosphates.
c. Buffer
d. Cofactors; Mg2+, Mn2+ and potassium ions
Properties of the polymerase:
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Isolated from Thermus aquaticus.
Heat stable (half-life 30min at 95c).
No proof reading function in 3’to 5’ direction.
Primer extension up to 100bases/sec.
Primer-dimer:
• Results from primers annealing
each other at 3’ end due to complementary
bases in the primers.
• Extended primers are no longer atcggactatcga
gctatacttatggcca
available to prime target for
PCR.
atcggactatcgatatgaataccgga
• Polymerase amplify the dimer
tagcctgatagctatacttatggcca
Primer-dimer:
Stages in PCR:
1. Denaturation:
Heat to separate ds (93-95c)
2. Annealing:
Primers bend to complementary seq
(50-70c).
3. Elongation:
adding of dNTPs.
Analysis of PCR products:
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Gel electrophoresis
Southern hyperdization
Restriction digestion
Denaturing gradient gel electrophoresis
DGGE: detect 95% of mutations
• Florescent PCR
Analysis of PCR products:
Florescent PCR:
• Primers labelled with florescent molecule
at 5’end
• Products detected by laser analysis system:
- exact sizing of PCR products
- can use more than one colour of florescent
ABI 310 Prism
Problems affecting PCR:
Problem
Possible reasons
No product
Primer annealing?
Product of incorrect size
Primer annealing else where in the
genome?
Several products formed
Contamination?
Several annealing sites?
PCR rxn inhibitors:
Proteinase K ---- digest polymerase
Phenol --------- denature the polymerase
Advantages of PCR:
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Uses less patient DNA
Quick results (3hrs)
Usually no radioactive materials
Precise in determining sizes of alleles
Detect point mutation
Less expensive
Limitations of PCR:
• Target DNA sequence must be known
• Errors of Taq-polymerase
• Size limitation (CG triplet repeats)
PCR based technologies:
1. Multiplex PCR: 1988
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Single template or multiple template
Different Pb length, to form distinct bands.
Target seq different enough.
Save time, effort
Cross hyperdization or miss-priming
2. QF-PCR;
Quantitative florescent PCR
• Primers tagged with florescent label;
Different colour & size (polymorphic markers).
• Used to know:
-If the seq is present or not
-No of copies in the sample
• Analysed by; automated genetic analyser.
QF PCR for prenatal diagnosis:
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Detect aneuploidy 13, 18, 21 & recently X chr.
CVS or amnio
results in less than 2 days
Looking for ratio 1:1 or 2:0 (normal)
Or 1:1:1, 2:1 or 3:0 (trisomy).
3.q-PCR; real time PCR:
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1996
amplified DNA is detected as the reaction progresses ‘real-time’
Uses: genotyping, mutation detection, gene expression
Instrumentation:
-ABI TaqMan:
96-well Block cycler with fluorimeter.
-Roche Lightcycler:
Glass capillary reaction tubes, 40 cycles in 15-20 min.
3.q-PCR; real time PCR:
**Compare sample by normalising
control
4- RT-PCR:
Reverse transcriptase PCR:
• Use mRNA as a template to produce cDNA;
Reverse transcriptase enzyme
• cDNA is then amplified to screen possible mutations directly.
Reverse transcriptase enzyme
Uses of RT-PCR:
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Diagnosis of genetic diseases (RNA).
Gene expression
Insertion of eukaryotic genes into prokaryotes
Studying the genomes of viruses composed of RNA;
Influenza virus A
HIV
Thank you
Questions?