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

G A A T G G C A C C A T T T A G C

C T T A C C G T G G T A A A T C G

PCR Properties:

Rapid & easy.Sensitive.Robust.Widespread applications;

Bioinformatics, forensics, medicine and genetic research.

PCR uses:

• Replacement of cloning.• Diagnosis of chromosomes abnormalities (QF-PCR).• Diagnosis of single gene defect.• Searching for genes and mutations.• Cancer genetics.

PCR requirements:

• 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:

• 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 complementarybases in the primers. • Extended primers are no longeravailable to prime target forPCR. • Polymerase amplify the dimer

atcggactatcga

gctatacttatggcca

atcggactatcgatatgaataccgga

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:

• 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 polymerasePhenol --------- denature the polymerase

Advantages of PCR:

• 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

• 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:

• 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:

• 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 enzymeRNA cDNA qPCR

Uses of RT-PCR:

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?