The polymerase chain reaction (PCR)

1. The polymerase chain reaction (PCR)

2. Experiment Goals • Understand how PCR technique works • Perform PCR experiment • Analyze PCR products

3. What is PCR? • Definition • The polymerase chain reaction (PCR) is a technique to amplify a piece of DNA very rapidly outside a living cell

4. Development of PCR • 1971: Khorana described basic principle of DNA replication using DNA primers • 1983: Dr. Karry Mullis developed PCR technique, for which he received the Nobel Prize in Chemistry in 1993.

5. PCR Applications • PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.

6. How does PCR work? • DNA is denatured (H-bonds are broken between strands of DNA with heat), 94oC • Primers attach to complementary sequences of single stranded DNA, 55-60oC • DNA polymerase attaches to primer with ssDNA and extends DNA fragment, 72oC • Thus, making double stranded DNA • This is done by changing the temperature

7. PCR Reaction Components 1) Target DNA - contains the sequence to be amplified. 2) Pair of Primers - oligonucleotides that define the sequence to be amplified. 3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks. 4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction 5) Mg++ ions - cofactor of the enzyme 6) Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme

8. 1) Target DNA • Target of DNA can be • a single gene • part of a gene • or a non-coding sequence

9. DNA Quality • DNA should be intact and free of contaminants that inhibit amplification. • Contaminants can be purified from the original DNA source. • Heme from blood, and melanin from hair • Contaminants can be introduced during the purification process. • Phenol, ethanol, sodium dodecyl sulfate (SDS) and other detergents, and salts.

10. DNA quantity • More template is not necessarily better. • Too much template can cause nonspecific amplification. • Too little template will result in little or no PCR product.

11. How Big A Target is? • Amplification products are typically in the size range 100-1500 bp. • Longer targets are amplifiable >25 kb. • Requires modified reaction buffer, cocktails of polymerases, and longer extension times.

12. 2) Pair of Primers • Primers define the DNA sequence to be amplified—they give the PCR specificity. • Primers bind (anneal) to the DNA template and act as starting points for the DNA polymerase, • DNA polymerases cannot initiate DNA synthesis without a primer. • The distance between the two primers determines the length of the newly synthesized DNA molecules.

13. 3) dNTPs (deoxynucleotidetriphosphates) • The building blocks for the newly synthesized DNA strands. • dATP, dGTP, dCTP or dTTP

14. 4) Thermostable DNA Polymerase • DNA Polymerase is the enzyme responsible for copying the sequence starting at the primer from the single DNA strand • Commonly use Taq, an enzyme from the hyperthermophilic organisms Thermus aquaticus, isolated first at a thermal spring • This enzyme is heat-tolerant • it is thermally tolerant (survives the melting T of DNA denaturation) • which also means the process is more specific, higher temps result in less mismatch – more specific replication

15. Running PCR • The PCR is commonly carried out in a reaction volume of 15-100 μl in small reaction tubes (0.2-0.5 ml volumes) in a thermal cycler. • The thermal cycler allows heating and cooling of the reaction tubes to control the temperature required at each reaction step. • Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. • Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. • Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.

16. Initialization step • Prior to the first cycle, there is an initialization step • the PCR reaction is often heated to a temperature of 94-96°C, and this temperature is then held for 1-9 minutes • This first hold is employed to ensure that most of the DNA template and primers are denatured, • Also, some PCR polymerases require this step for activation

17. PCR Reaction Cycles • One PCR cycle consists of a DNA denaturation step, a primer annealing step and a primer extension step. • DNA Denaturation: Expose the DNA template to high temperatures to separate the two DNA strands and allow access by DNA polymerase and PCR primers. • Primer Annealing: Lower the temperature to allow primers to anneal to their complementary sequence. • Primer Extension: Adjust the temperature for optimal thermostable DNA polymerase activity to extend primers.

18. PCR

19. PCR: First 4 Cycles

20. PCR: Completed Amplification Cycle

21. PCR: Completed Amplification Cycle • Each cycle: 1 copy of DNA template will give 2 copies from double-stranded DNA templates. • n cycles will give 2ncopies • Assuming a cycle lasts 6 min: • 1 double-stranded DNA molecule • 35 cycles • 34x109 copies in 3.5 hrs! • There are also ≈ 60 other DNA copies

23. Analyze PCR products • Check a sample by gel electrophoresis. • Is the product the size that you expected? • Is there more than one band? • Is any band the correct size? • May need to optimize the reaction conditions.

24. PCR Modifications • Nested PCR • increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are being used in two successive PCRs. • Multiplex PCR • The use of multiple, unique primer sets within a single PCR mixture to produce amplicons of varying sizes specific to different DNA sequences. • Reverse-transcriptase PCR • (Reverse Transcription PCR) is a method used to amplify, isolate or identify a known sequence from a cellular or tissue RNA. The PCR is preceded by a reaction using reverse transcriptase to convert RNA to cDNA.

25. Polymerase Chain ReactionControls for PCR • Blank reaction (Negative control reaction) • Controls for contamination • Contains all reagents except DNA template • Positive control reaction • Controls for sensitivity • Contains all reagents and a known target-containing DNA template

26. Contamination of PCR Reactions • Most common cause is carelessness and bad technique. • Separate pre- and post-PCR facilities. • Dedicated pipettes and reagents. • Change gloves. • Aerosol barrier pipette tips. • 10% bleach, UV light

27. Procedure 1- Prepare master Mix 2- Program the thermocycler 3- Run the samples on thermocycler 4- Analysis of PCR products

28. Target DNA • Amplification of part of the Human growth hormone gene • Specific primers used • Forward primer: 5’- TCCCTTCCCAACCATTCCCTTA-3’ • Reverse primer: 5’-CCACTCACGGATTTCTGTTGTGTTTC-3’

29. 1- Master Mix

30. 2- Program the Thermocycler • The Thermocycler Profile is: • Step 1: Denaturation for 3 min. at 95oC • Step 2: 35 cycles • Melting for 60 sec. at 95oC • Annealing for 60 sec. at 57oC • Extension for 90 sec. at 72oC • Step 3: Final elongation for 10 min. at 72oC

31. 4- Analysis of PCR products • Analyse products on 2% agarose gel containing ethidium bromide • Visualize the PCR product on UV transilluminator +ve -ve Sample