Polymerase chain reaction and it’s modifications

PCR & it's Modifications.
This presentation contains basic PCR process and it's 4 important modifications.

1. Polymerase Chain Reaction & It’s Modifications Hot Start PCR Q-PCR RT-PCR Nested PCR Kary Mullis

2. Polymerase Chain Reaction PCR is technology in molecular biology used to amplify a single or a few copies of DNA across several orders generating thousands and millions copies of a particular sequence. PCR takes a specific sequence of DNA of small amounts and amplifies it to be used for further testing. It relies on thermal cycling consisting of cycles of repeated heating & cooling of the reaction for DNA melting and enzymatic replication of the DNA.

3. Processes Denaturation - It is the first step where DNA stands are separated by heating At 950 C. Annealing - It is the process in which two stands of DNA are allowed to form hydrogen bonds. (550 C – 650 C) Extension- The process in which the nucleotides are added to primer by Taq polymerase. (720 C)

4. The basic protocol—what’s in the tube Target DNA 5 ’ 3 ’ 3 ’ 5 ’ Primers (0.1-o.5uM) A B Free Nucleotides (20-200uM) Taq DNA Polymerase 1-2.5units Mg 2 + Mg 2 + Mg 2 + Mg 2 + Mg 2 + Mg 2 + Buffer containing magnesium (MgCl2) 0.5-2.5mM

5. The basic protocol--denaturation Target DNA 95 o C 5 ’ 3 3 ’ 5 5 ’ 3 ’ 3 ’ 5

6. The basic protocol--annealing ~ 55 o C 5 3 ’ 3 ’ 5 ’ 5 ’ 3 3 ’ 5 ’ 5 ’ 5 ’ Target DNA A B primers A B

7. The basic protocol--extension 72 o C 5 ’ 3 3 ’ 5 ’ 5 ’ 3 ’ 5 5 ’ 5 ’ Target DNA Taq polymerase 3 ’

8. The basic protocol--extension 72 o C ’ 5 3 ’ 5 5 ’ 3 3 5 ’ 5 ’ 5 ’ Target DNA 3 ’

9. MODIFICATIONS OF PCR HOT-START PCR QUANTITATIVE PCR REVERSE TRANSCRIPTION PCR NESTED PCR

10. HOT-START PCR It is a modified form of PCR which avoids non-specific amplification of DNA by inactivating Taq polymerase at lower temperature In the second step in addition to primer and Taq polymerase we add specific antibodies to block Taq polymerase from annealing. When temperature raises for amplification at 72o C, the specific antibodies detaches from Taq polymerase & amplification begins with greater specificity.

11. HOT-START PCR

12. Q-PCR (Quantitative-PCR) It is also known as Real Time PCR Real Time PCR is a laboratory technique of molecular biology based on the PCR, which is used to amplify & simultaneously detect or quantify targeted DNA molecules. This is the new approach compared to std. PCR where the product of the reaction is detected at it’s end.

13. Methods Non specific fluorescent dyes that intercalate with any dsDNA. Sequence specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent with a fluorescent reporters which permits detection only after hybridization of probe with it’s complimentary sequence to quantify mRNA or non coding RNA in cells or tissue.

14. TaqMan Probe

15. Fluorescent dyes

16. Reverse Transcription PCR In RT PCR, the RNA templates first converted to complementary DNA ( cDNA ) using reverse transcriptase. The cDNA is used as template for amplification of DNA by PCR The amplification of RNA can be done with the help of ONE STEP PCR & TWO STEP PCR

17. NESTED PCR Nested polymerase chain reaction (PCR) is a modification of PCR intended to reduce the contamination in products due to the amplification of unexpected primer binding sites. Nested PCR utilizes two different sets of primers during a two-step amplification. The PCR reaction is run using the "outer primers“ during a first cycle of amplification which is immediately followed by a second cycle of amplification carried out with the "inner primers".

18. NESTED PCR NESTED PCR is a variation of the PCR, using two pairs of primers to amplify fragments of DNA. The first pair primer amplify a fragment similar to standard PCR. The second pair of primers called nested primers (because they are in the first PCR amplification of an internal fragment) incorporated inside the first PCR product, so that the second PCR amplified fragment is shorter than the first amplification.