PCR

Polymerase chain reaction

Transcript

1. BY MALEEHA FATIMA
2. What is PCR? The polymerase chain reaction (PCR) is a rapid, inexpensive, popular molecular biology technique for enzymatically replicating DNA (without using a living organism such as E.coli or yeast) & produces microgram amounts of DNA from minute quantities of template. This technique allows a small amount of the DNA molecule to be amplified many times in an exponential manner.
3. History PCR technique was developed in 1983 by Kary Mullis. In 1993, Mullis was awarded the Nobel prize in Chemistry along with Michael Smith for his work on PCR. Dr. Kary Mullis stated it “lets you pick the piece of DNA you are interested in & have as much of it as you want”.
4. Steps The PCR process consists of a series of twenty to thirty-five cycles. Each consists of three steps: 1. Denaturation of dsDNA 2. Annealing of the primers 3. Extension of the primers
5. The double-stranded DNA has to be heated to 94-96 °C in order to separate the strands. This is called denaturing; it breaks apart the hydrogen bonds that connect the two DNA strands. Prior to the first cycle, the DNA is often denatured for an extended time to ensure that both the template DNA & the primers have completely separated & are now single-strand only. Time 1-2 minutes up to 5 minutes. Also Taq–polymerase is activated by this step. Denaturing
6. After separating the DNA strands, the temperature is lowered so the primers can attach themselves to the single DNA strands. This step is called annealing. The temperature of this stage depends on the primers & usually 50 °C below their melting temperature (45-60 °C). A wrong temperature during the annealing step can result in primers not binding to the template DNA at all, or binding at random. Time 1-2 minutes. Annealing
7. Extension Finally, the DNA-polymerase has to fill in the missing strands. It starts at the annealed primer & works its way along the DNA strand. This step is called extension. The extension temperature depends on the DNA-polymerase. The time for this step depends both on the DNA-polymerase itself & on the length of the DNA fragment to be amplified. As a rule-of-thumb, 1minute per 1 kbp.
8. Stages The PCR process can be divided into three stages: 1. Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). This reaction is very sensitive, only minute quantities of DNA need to be present. 2. Leveling off stage: The reaction slows as the DNA polymerase loses activity & as consumption of reagents such as dNTPs & primers causes them to become limiting. 3. Plateau: No more product accumulates due to exhaustion of reagents & enzymes.
9. Components • DNA template or cDNA which contains the region of the DNA fragment to be amplified • Two primers which determine the beginning & end of the region to be amplified • Taq polymerase (DNA polymerase) extends primers & copies the region to be amplified • Nucleotides from which the DNA-polymerase for new DNA • Buffers provides a suitable chemical environment for the DNA-polymerase • Mg²+ is required as co-factor for the thermostable DNA polymerase • Gelatin & Triton X-100 stabilise the DNA polymerase • dNTPs provide initial excess required for incorporation into DNA
10. PRIMERS These are short, artificial DNA strands not more than fifty, usually 18-25 bp nucleotides that are complementary to the beginning & end of the DNA fragment to be amplified. Both primers which used in the reaction, should have similar annealing temperatures with a minimal degree of self-complementarity in order to avoid the formation of secondary structures & no complementarity to each other so that the primers dimers will not formed.
11. The PCR reaction is carried out in a machine that heats & cools the reaction tubes within it, to the precise temperature required for each step of the reaction, is called Thermal Cycler.
12. The PCR product can be identified by its size using agarose gel electrophoresis.
13. Applications ✓ Diagnosis & screening of genetic diseases & cancer ✓ Rapid detection of slowly growing microorganisms & viruses ✓ HLA typing in transplantation ✓Analysis of DNA in archival material ✓ DNA fingerprinting in forensic science ✓ Preparation of nucleic acid probes ✓ Clone screening, mapping & sub-cloning ✓ Paternity test
14. Disadvantages ▪ requires costly instruments ▪ adequate space with aircondition, dehumidifier, laminar flow facilities ▪ costly & not all people can afford to do test ▪ requires trained, experienced, qualified manpower & technologists ▪ false positive & false negative results may lower specificity & sensitivity ▪ limited scope for diagnosis of diseases
15. Practical Modification These are some practical modifications to PCR technique: • Nested PCR • Inverse PCR • Reverse Transcriptase RT-PCR • Asymetric PCR • Quantitative PCR • Real time PCR • Touchdown PCR • Colony PCR • Allele-specific PCR
16. • After-The-Exponential-PCR • Dial-out PCR • Helicase-dependent amplification • Hot start PCR • Intersequence-specific PCR • Ligation-mediated PCR • Methylation-specific PCR • Miniprimer PCR • Assembly PCR • Multiplex PCR • Amplification fragment length polymorphism (AFLP)
17. • Arbitrary Primed PCR • In-situ PCR • Overlap-extension PCR • Solid phase PCR • Universal fast walking • Variable number of tandem repeats (VNTR) PCR • Long accurate PCR • High fidelity PCR • Fast PCR • GC-Rich PCR • Long-range PCR