Dr. Sumbul FatmaDepartment of Medical

Biochemistry

What is PCR?It’s a means of selectively amplifying a particular

segment of DNAEach cycle of amplification doubles the amount of

DNA in the sampleSource of DNA could be any- bacterial, viral, plant

and animal

Dr. Sumbul Fatma

Advantages of PCR?PCR allows the DNA in a single cell, hair follicle, or

spermatozoan to be amplified and analyzedDNA sequences as short as 50-100bp and as long

as 10kb can be amplifiedAs few as 20 cycles would yield ~106 times the

amount of target DNA initially present

Dr. Sumbul Fatma

The invention of PCR

Invented by Kary B Mullis in 1983

First published account appeared in 1985

Awarded Nobel Prize for Chemistry in 1993

Dr. Sumbul Fatma

Requirements of a PCRDNA polymerase- to repetitively amplify targeted portion

of DNANucleotide triphosphates- ATP, GTP, CTP and TTPPrimers- two single stranded oligonucleotides (20-25ntds

long), which are complimentary to the flanking sequences that bracket the target DNA sequence

Dr. Sumbul Fatma

Thermal Cycler

PCR cyclers are available from many suppliers

Reactions are done in tubes or 96 well microtitre plates

Dr. Sumbul Fatma

Requirements of a PCR

Steps of a PCRPrimer construction- it is synthetic oligonucleotide

complimentary to the short nucleotide segments on each side of the target DNA

Dr. Sumbul Fatma

Steps of a PCRDenature the DNA- The DNA to be amplified is

heated to separate the double stranded target DNA into single strands(1 min. 940C )

Dr. Sumbul Fatma

Steps of a PCRAnnealing of primers to ssDNA- the separated

strands are cooled and allowed to anneal to the two primers (one for each strand)

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45 sec, 540CForward and reverse primers

Steps of a PCRChain extension- the DNA polymerase adds

nucleotides to the 3’-hydroxyl end of the primer, and strand growth extends across the target DNA, making complimentary copies of the target (2 min 720C)

At the completion of one cycle of replication, the reaction mixture is heated again to denature the DNA strand (of which there are now 4)

Dr. Sumbul Fatma

Polymerase Chain Reaction

ThermocyclingDenaturation - 940CAnnealing - 550CExtension - 720CDenaturation again………….20-30 cyclesThe amplified target sequence is called ampliconsWith each cycle there is an exponential increase in

the amount of target DNA, hence the name “Polymerase Chain Reaction”

Dr. Sumbul Fatma

DNA polymerase in PCR

Dr. Sumbul Fatma

Heat stable DNA polymerase is vital to the ease of the process ……

Dr. Sumbul Fatma

Thermus aquaticus, a thermophilic bacteria that lives and replicates at 70-800C is the source of Taq DNA polymerase used in PCR reactions.

Multiple cycles of PCR

Dr. Sumbul Fatma

The target is RNA ?the RNA must be enzymatically converted to DNAReverse Transcriptase- are the RNA directed DNA

polymerases Reverse Transcriptase

RNA cDNAcDNA is then amplified by PCR

This process is termed as RT-PCR

Dr. Sumbul Fatma

Analysis of PCR productsAmplicons can be analyzed by gel electrophoresis

and Southern Blot- qualitative analysis

Quantitative PCR- used to measure the viral loads in HIV and Hep C-infected patients

These numbers allow physicians to determine disease status and evaluate efficacy of antiviral treatment.

Dr. Sumbul Fatma

Real Time PCRDoes not measure the amount of end product of

PCR but its production or accumulation in real timeTwo common methods of quantification are-

1. the use of fluorescent dyes that intercalate with double-stranded DNA e.g. SYBR green

2. modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA (Taqman probes, molecular beacons and scorpion primers)

Dr. Sumbul Fatma

Real Time PCR- detection methodsFluorescent dyes like SYBR green-

A DNA-binding dye binds to all dsDNA in PCR, causing fluorescence of the dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence intensity and is measured at each cycle, thus allowing DNA concentrations to be quantified

Dr. Sumbul Fatma

Real Time PCR- detection methods

Dr. Sumbul Fatma

Unhybridized probe has donor fluorophore and non-fluorophore acceptor molecule (quenchers) in close proximity – no signal • Upon hybridization to the target, the fluorophore and quencher become separated through either

•Conformational change- molecular beacons, scorpion primers•Enzymatic cleavage of the fluorophore from the quencher as a result of 5’ to 3’ nuclease activity of the Taq DNA polymerase- Taqman probes

Applications of PCR

Dr. Sumbul Fatma

Comparison of a normal cloned gene with an uncloned mutant form of the gene

Detection of low abundance nucleic acid sequences e.g. viruses, mRNA in cells or tissue

Forensic analysis of DNA samplePrenatal diagnosis and carrier detection of Cystic

Fibrosis

Cystic FibrosisIt is an autosomal recessive

disorderResults from mutations in the

cystic fibrosis transmembrane conductance regulator gene

The most common mutation is loss of Phe residue from the protein

Distinguished by difference in size of the mutated PCR product

Dr. Sumbul Fatma

ReferencesLippincott ‘s Illustrated Reviews, 4th EditionClinical Chemistry: Principles, Procedures,

Correlations by Michael L Bishop

Dr. Sumbul Fatma