1. 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. Sumbul Fatma

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 ~10 6 times the amount of target DNA initially present Dr. Sumbul Fatma

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. Sumbul Fatma

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. Sumbul Fatma

6. 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

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. Sumbul Fatma

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. 94 0 C ) Dr. Sumbul Fatma

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) Dr. Sumbul Fatma 45 sec, 54 0 C Forward and reverse primers

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 72 0 C) 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

11. Polymerase Chain Reaction

12. Thermocycling Denaturation - 94 0 C Annealing - 55 0 C Extension - 72 0 C 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. Sumbul Fatma

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

14. Taq DNA polymerase in PCR Dr. Sumbul Fatma Thermus aquaticus, a thermophilic bacteria that lives and replicates at 70-80 0 C is the source of Taq DNA polymerase used in PCR reactions.

15. Multiple cycles of PCR Dr. Sumbul Fatma

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 as RT-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. Sumbul Fatma

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. Sumbul Fatma

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. Sumbul Fatma

20. 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

21. 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 sample Prenatal diagnosis and carrier detection of Cystic Fibrosis

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. Sumbul Fatma

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