PCR presentation

Pcr presentation

1. PCR (POLYMERASE CHAIN REACTION) GROUP MEMBERS HIBA AKHTAR(SP18-BSI-024) KASHAF NAZ(SP18-BSI-025)

2. Table of contents: What is PCR? Short history of PCR Principle of PCR Purpose of PCR Components of PCR Procedure of PCR Variants of PCR Applications of PCR Problems and limitations with PCR Conclusion

3. What is PCR? PCR stands for polymerase chain reaction. It is the technique widely used in molecular biology to make several copies of a specific DNA segment PCR is a technique that takes specific sequences of DNA of small amount and amplifies it to be used in further testing. Makes millions of copies of a particular segment of DNA in a given time span In vitro technique

4. Short history of PCR 1983: Dr.kary mullis developed PCR 1985: first publication of PCR by cetus corporation appears in science. 1986: purified Taq polymerase was first used in PCR. 1988: PerkinElmer introduces the automated thermal cycler. 1989: science declares Taq polymerase “molecule of the year” 1990: amplification and detection of specific DNA sequence using a fluorescent DNA-binding dye, laying the foundation for future “real-time” or “kinetic” PCR 1991: RT-PCR for diagnostic tests for RNA viruses was discovered using single thermo stable polymerase.

5. Purpose of PCR The purpose of the polymerase chain reaction is to amplify a lot of double-stranded DNA molecules (fragments) with same length having same (identical)sizes and sequences by using the enzymatic method and the process is in the form of cyclic conditions

6. Principle of PCR PCR involves the primer mediated enzymatic amplification of DNA . PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand. Primer is needed because DNA polymerase can add a nucleotide only onto a preexisting 3’-OH group to add the first nucleotide . DNA polymerase then elongate its 3 end by adding more nucleotides to generate an extended region of double stranded DNA

7. Components of PCR The PCR technique requires the following components: DNA template : The double stranded DNA (ds DNA) of interest , separated from sample. DNA polymerase : usually a thermo stable Taq polymerase that does not rapidly denature at high temperature (98 degrees) , and can function at a temperature optimum of about 70 degree centigrade Oligonucleotide primers : short pieces of single stranded DNA (often 20-30 base pairs) which are complementary to 3’ ends of the sense and the antisense strands of the target sequence.

8. 4. Deoxynucleotide triphosphate : single units of bases A, T,C,G (d ATP , d TTP , d GTP ,d CTP) provide the energy for polymerization and the building blocks for DNA synthesis. 5. Buffer system : includes magnesium potassium to provide the optimal conditions for DNA denaturation and renaturation ; also important for polymerase activity , stability and fidelity.

9. Procedure of PCR The process of PCR is start by mixing all the PCR components together and taken through the series of 3 major cyclic reactions , conducted in an automated , self-contained thermo cycler machine . The all important feasible conditions are very crucial to be provided to carry out the procedure of polymerase chain reaction efficiently.

10. Denaturation This step involves heating the reaction mixture to 94 degree centigrade for the duration of 15-30 seconds. During this process , the double stranded DNA is denatured to single strands due to breakage in weak hydrogen bonds.

11. Denaturatin process

12. Annealing the reaction temperature is rapidly lowered to 54-60 degree centigrade for the time span of 20-40 seconds not longer of that . This step allows the primers to bind (anneal) to the complementary sequence in the template DNA . So that the procedur of PCR can begin.

13. Annealing process

14. Elongation This step is also known as extension , this step usually occurs at 70-80 degree centigrade (most commonly 72 degree centigrade) .72 degrees is the optimum temperature for the Taq polymerase to built the complementary strand .it attaches to the primer and then adds DNA bases to the single strand one-by-one in 5’ to 3’ direction. DNA polymerase will add about 1,000 bp /minute

15. Elongation process

16. Process of PCR

17. Variants of PCR Standard PCR : sequences both ends of target DNA have to be known . Two primers define the ends of target DNA and only that part is amplified. Single sided PCR : here DNA is rearranged before amplification so that only one primer is needed .this is also called Anchored PCR. Inverse PCR : DNA at primer sites rather than between two primers is amplified because primer sites which are bracketing may have important sequence like promoter for triggering target gene into action.

18. Applications of PCR PCR is used in cloning . PCR is also being widely used in forensic DNA detection. PCR technique can also be used to identify the transgenic plants. PCR can be used for the detection of viral infections. PCR is used in the detection of ancient DNA for the evolutionary study purposes. PCR in comparative study of genomics. PCR is also used in gene manipulation and expression study. PCR in DNA sequencing.

19. Problems and limitations with PCR Contamination of the reaction mixture by bacteria, viruses and our own DNA presents a real problem. PCR can not substitute for a cell-based gene cloning, when large amounts of gene are desired. Taq polymerase used in PCR often lacks 3’ to 5’ exonuclease activity .this enzyme lacks the ability to correct mis-incorporated nucleotides. PCRs of longer products are less efficient due to enzyme activity loss . Applies only to short DNA fragments.

20. conclusion PCR is not only vital in clinical laboratories by amplifying small amounts of DNA for STD detection but also important for genetic predisposing for defects such as factor V Leiden. The PCR technology can also be employed in law enforcement ,genetic testing of animal stocks and vegetable hybrids , and drug screening along with many more areas. PCR is a very important time saving technique to make a huge number of copies of a specific sequence of DNA required.