Polymerase Chain Reaction (PCR)

1. Polymerase Chain Reaction (PCR) Dr. Sumbul Fatma Department of Medical Biochemistry

2. What is PCR? • It’s a means of selectively amplifying a particular segment of DNA • Each cycle of amplification doubles the amount of DNA in the sample • Source of DNA could be any- bacterial, viral, plant and animal Dr. SumbulFatma

3. Advantages of PCR? • PCR allows the DNA in a single cell, hair follicle, or spermatozoan to be amplified and analyzed • DNA sequences as short as 50-100bp and as long as 10kb can be amplified • As few as 20 cycles would yield ~106 times the amount of target DNA initially present Dr. SumbulFatma

4. 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. SumbulFatma

5. Requirements of a PCR • DNA polymerase- to repetitively amplify targeted portion of DNA • Nucleotide triphosphates- ATP, GTP, CTP and TTP • Primers- two single stranded oligonucleotides (20-25ntds long), which are complimentary to the flanking sequences that bracket the target DNA sequence Dr. SumbulFatma

6. Requirements of a PCR Thermal Cycler • PCR cyclers are available from many suppliers • Reactions are done in tubes or 96 well microtitre plates Dr. SumbulFatma

7. Steps of a PCR • Primer construction- it is synthetic oligonucleotide complimentary to the short nucleotide segments on each side of the target DNA Dr. SumbulFatma

8. Steps of a PCR • Denature the DNA- The DNA to be amplified is heated to separate the double stranded target DNA into single strands(1 min. 940C ) Dr. SumbulFatma

9. Steps of a PCR • Annealing of primers to ssDNA- the separated strands are cooled and allowed to anneal to the two primers (one for each strand) 45 sec, 540C Forward and reverse primers Dr. SumbulFatma

10. Steps of a PCR • Chain 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. SumbulFatma

11. Polymerase Chain Reaction

12. Thermocycling • Denaturation - 940C • Annealing - 550C • Extension - 720C • Denaturation again…………. • 20-30 cycles • The amplified target sequence is called amplicons • With each cycle there is an exponential increase in the amount of target DNA, hence the name “Polymerase Chain Reaction” Dr. SumbulFatma

13. DNA polymerase in PCR Heat stable DNA polymerase is vital to the ease of the process …… Dr. SumbulFatma

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

15. Multiple cycles of PCR Dr. SumbulFatma

16. The target is RNA ? • the RNA must be enzymatically converted to DNA • Reverse Transcriptase- are the RNA directed DNA polymerases Reverse Transcriptase RNA cDNA • cDNA is then amplified by PCR • This process is termed asRT-PCR Dr. Sumbul Fatma

17. Analysis of PCR products • Amplicons 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. SumbulFatma

18. Real Time PCR • Does not measure the amount of end product of PCR but its production or accumulation in real time • Two 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. SumbulFatma

19. Real Time PCR- detection methods • Fluorescent 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. SumbulFatma

20. Real Time PCR- detection methods • 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 Dr. SumbulFatma

21. Applications of PCR • 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 sample • Prenatal diagnosis and carrier detection of Cystic Fibrosis Dr. SumbulFatma

22. Cystic Fibrosis • It is an autosomal recessive disorder • Results 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. SumbulFatma

23. References • Lippincott ‘s Illustrated Reviews, 4th Edition • Clinical Chemistry: Principles, Procedures, Correlations by Michael L Bishop Dr. Sumbul Fatma