Polymerase Chain Reaction

Introduction, Evolution of PCR, Principle, Instrumentation, Reagents, Primer Designing, DNA polymerase, Components of PCR, PCR Types, Applications

1. POLYMERASE CHAIN REACTION Advanced Pharmaceutical Analysis Presented by- Mehul H Jain M. Pharmacy I-I Pharmaceutical Analysis

2. Contents Introduction Evolution of PCR Principle Instrumentation Reagents Primer Designing DNA Polymerases Components of PCR PCR types Application

3. Introduction

4. DNA Replication Process of producing two identical copies of DNA from original DNA molecule.

5. PCR PCR, polymerase chain reaction, is an in-vitro technique for amplification of a region of DNA whose sequence is known or which lies between two regions of known sequence. Before PCR, DNA of interest could only be amplified by over-expression in cells and this with limited yield. Polymerase Chain Reaction (PCR) - amplify a known sequence of DNA to several orders of magnitude. Chain reaction - Small DNA fragment of interest acts as template for the synthesis of new DNA strand. One DNA molecule is used to produce multiple copies exponentially. DNA polymerase will add nucleotides to the free 3-OH of this primer according to the normal base pairing rules. PCR typically amplifies DNA fragments of between 0.1 and 10 kilo base pairs.

6. Exponential Amplification

7. Evolution Of PCR

8. 1971 - Gobind Khorona described replication of short fragments of DNA using primers & polymerases in-vitro. 1983 - Kary Mullis invented PCR. 1986 - Purified Taq Polymerase first used in PCR. 1976 - Isolation of DNA polymerase from Thermus aquaticus. 1985 - Mr. Cycler was invented. 1988 - Perkin Elmer introduced the automated thermo cycler.

9. Principle

10. PCR consists of a series of 20 - 40 cycles. One PCR cycle comprises of 3 steps Denaturation   Annealing Extension Temperatures used and the length of time applied in each cycle depends on T m of primers, DNA polymerase, dNTPs & divalent ion concentration. Initialization Step: In this step the reaction is heated to 94-96°C for 30 seconds to several minutes. This step is usually only done in the very beginning of the PCR reaction. This step is important for activating hot-start polymerases, if we use such a polymerase and to denature your template DNA. If the template GC content is high we may need to perform an extra-long initialization step. Heating dissociates the inhibitor - DNA polymerase complex.

11. Step – I: Denaturation First cycling event. Heating reaction mixture 94 - 96 °C for 30 seconds. DNA melting by disrupting the hydrogen bonds between bases yields single stranded DNA. Step – II: Annealing Reaction temperature is lowered to 50 - 65°C for 20 - 40 seconds. The temperature in this step needs to be low enough that the denatured primers can form Watson-Crick base pairs with the template DNA. But high enough that only the most stable (perfectly paired) double-stranded DNA structures can form. Usually this perfect annealing temperature is a few degrees lower than the melting temperature of the primer pair. During this step the polymerase will binds to the primer/template DNA complex. Although the polymerase will not start reading until the temperature is raised in the next step.

12. Step – III: Extension Temperature depends on polymerase used.  Taq polymerase has its optimum activity temperature at 75 - 80°C. DNA polymerase synthesizes new DNA strand complementary to the DNA template strand by adding dNTPs. Extension time depends on DNA polymerase used as well as length of the DNA fragment to amplify. At its optimum temperature, the DNA polymerase polymerizes a thousand bases per minute.

13. PCR Steps

14. Reagents & Instrumentation

15. PCR Requirements DNA template containing desired segment Primers DNA polymerase Deoxy nucleoside triphosphates dNTPs. Buffers Bivalent cations – Mg 2+ , Mn 2+ Monovalent cation - K + Additives

16. Thermal Cycler Earliest thermal cyclers were designed for use with the Klenow fragment of DNA polymerase I. Peltier element - Modern PCR machines. Lid temperature - 105 o C. Thermal blocks - 48/96 wells. The thermal cycler (also known as a thermocycler, PCR machine or DNA amplifier) is a  laboratory  apparatus most commonly used to amplify segments of  DNA  via the  polymerase chain reaction  (PCR). Thermal cyclers may also be used in laboratories to facilitate other temperature-sensitive reactions, including  restriction enzyme  digestion or rapid diagnostics. The device has a  thermal block  with holes where tubes holding the reaction mixtures can be inserted. The cycler then raises and lowers the temperature of the block in discrete, pre-programmed steps.

17. With one cycle, a single segment of double-stranded DNA template is amplified into two separate pieces of double-stranded DNA. These two pieces are then available for amplification in the next cycle. As the cycles are repeated, more and more copies are generated and the number of copies of the template is increased exponentially.

18. Primer Designing

19. Primer Primer is an oligonucleotide sequence – 18-26 bp in length provides free 3’-OH for the attachment of nucleotide bases by Polymerase.  Primers need to match the beginning and the end of the DNA fragment to be amplified. In PCR, both the strands will be amplified. So, one primer each for both the strands must be designed.  Forward primer - beginning of gene of interest.  Reverse primer beginning of complementary strand (in the 5' end).

20. Primer Length : Optimal length of PCR primers is 18-26 bp.  Long enough for adequate specificity and short enough for primers to bind easily to the template. Primer Secondary Structures: Intermolecular or intramolecular interactions creates primer secondary structures leads to poor or no yield of the product. Affects primer template annealing and thus the amplification. Primer Melting Temperature: Temperature at which one half of the DNA duplex will dissociate to become single stranded. Primers with melting temperatures in the range of 52-58 o C produce the best results.  GC content of the sequence gives a fair indication of the primer T m .

21. Annealing Temperature (Ta):   Annealing temperature (Ta) relies directly on length and composition of the primers.  Ta must be set 5 o C below the Tm of your primers.  High T a - insufficient primer-template hybridization. low T a - non-specific binding. GC Content:  GC content of the primer should be 40-60%. GC Clamp:  Presence of G or C bases within the last five bases of primers (GC clamp) promote specific binding. More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the primer.

22. DNA Polymerases

23. Taq Polymerase: Thermophilic bacterium - Thermus aquaticus.   Optimum temperature – 70 - 80°C. Half-life - 40 minutes at 95°C.  Lacks 3’ – 5’ proofreading activity. 1 error in every 10 4 nucleotides incorporated. pfu DNA Polymerase:   Isolated from Pyrococcus furiosus.   3’ - 5’ & 5’ - 3’ exonuclease activity.  Fidelity of enzyme is 12 fold higher.  Half life at 95°C - 2 hours.

24. Components of PCR

25. The PCR reaction requires the following components: DNA Template : The double stranded DNA (dsDNA) of interest, separated from the sample. DNA Polymerase : Usually a thermostable Taq polymerase that does not rapidly denature at high temperatures (98°), and can function at a temperature optimum of about 70°C. Oligonucleotide primers : Short pieces of single stranded DNA (often 20 - 30 base pairs) which are complementary to the 3’ ends of the sense and anti- sense strands of the target sequence. Deoxynucleotide triphosphates : Single units of the bases A, T, G, and C (dATP, dTTP, dGTP, dCTP) provide the energy for polymerization and the building blocks for DNA synthesis. Buffer system : Includes magnesium and potassium to provide the optimal conditions for DNA denaturation and renaturation; also important for polymerase activity, stability and fidelity.

26. Types of PCR

27. Reverse Transcriptase - PCR:  Detect gene expression through the synthesis of complementary DNA (cDNA) transcripts from RNA.  RNA template converted into a complementary DNA (cDNA) using a reverse transcriptase.  Primers - Oligo dt, random primers & gene specific primers. One step & Two step RT-PCR.  Amplified DNA fragments that are produced can by analyzed by agarose gel electrophoresis.  Amount of amplified fragment produced proportional to the amount of target mRNA in the original RNA sample.

28. Real Time PCR or (q-PCR):   In RT-PCR, process of amplification of DNA is monitored in real time.  PCR with an added probe or dye to generate a fluorescent signal from the product.  Detection of signal in real time allows quantification of starting material.  Performed in specialized thermal cyclers with fluorescent detection systems.  PCR signal is observed as an exponential curve with a lag phase, a log phase, a linear phase, and a stationary phase.

29. Assembly PCR:   Formation of large oligo nucleotides of DNA from short segments. Each oligonucleotide is designed to be either part of the top or bottom strand of the target sequence.  Oligonucleotides anneal to complementary fragments and then are filled by polymerase. Each cycle thus increases the length of various fragments randomly depending on which oligonucleotides find each other.  Production of synthetic genes and even entire synthetic genomes.

30. Asymmetric PCR:  Amplifies one strand of the target DNA.  Used in sequencing and hybridization probing - amplification of only one of the two complementary strands is required. PCR is carried out with a great excess of primer for the strand targeted for amplification.

31. Colony PCR:  Screening of bacteria (E.coli) or yeast clones for correct ligation or Plasmid products. Individual transformants can either be lysed in water with a short heating step or added directly to the PCR reaction and lysed during the initial heating step.  Initial heating step causes the release of the plasmid DNA from the cell, so it can serve as template for the amplification reaction.  Primers designed to specifically target the insert DNA can be used to determine if the construct contains the DNA fragment of interest and also insert orientation. Hot-start PCR Inverse PCR Nested PCR Touch down PCR

32. Applications

33. Cystic Fibrosis (CF): CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator ( CTFR ) gene.  In non-CF individuals, the CTFR gene codes for a protein that is a chloride ion channel and is involved in the production of sweat, digestive juices and mucus.  In CF individuals, mutations in the CTFR gene lead to thick mucous secretions in the lungs and subsequent persistent bacterial infections.  The presence of CTFR mutations in a individual can be detected by performing PCR and sequencing on that individual’s DNA. Human Immunodeficiency Virus (HIV): HIV tests rely on PCR with primers that will only amplify a section of the viral DNA found in an infected individual’s bodily fluids.  Therefore if there is a PCR product, the person is likely to be HIV positive. If there is no PCR product the person is likely to be HIV negative.

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