Rajan sharma

 Polymerase chain reaction Is a in vitro method of enzymatic synthesis of specific DNA sequences.  This method was first time developed by Kary Mullis in  It is a very simple and inexpensive technique for characterizing, analyzing and synthesizing any specific piece of DNA or RNA from virtually any living organism.

 PCR works on the basic principle of three temperature cycles: 1. Denaturation(92°c-96°c)-two strands melt open to form ssDNA. 2. Annealing(45°c -55°c)-annealing of primers to each original strand for new strand synthesis. 3. Extension.(72°c)-polymerase adds dNTPs complementary to the template at the 3’ end of the primers, since both strands are copied during PCR there is an exponential in in the no. of copies of the genes.

 These three steps are repeated times in an automated thermo cycler that can heat and cool the reaction mixture in tubes within a very short time.  Amplification can be calculated as per following formula: Amplification=2n, where n=no of cycles

1. DNA 2. PCR  Targets  Denaturing  Primers  Annealing  Cycles  Requirements

3. Applications of PCR  Neisseria gonorrhea  Chlamydia  HIV-1  Factor V Leiden  Forensic testing 4. Extraction of DNA for Factor V 5. DNA Detection for Factor V 6. PCR Results for Factor V 7. Conclusion

 DNA is a nucleic acid that is composed of two complementary nucleotide building block chains.  The nucleotides are made up of a phosphate group, a five carbon sugar, and a nitrogen base.  Generally nanogram or picogram amount of DNA is used.  Higher amount of DNA inhibits or results in non specific amplification.

 The targets in PCR are the sequences of DNA on each end of the region of interest, which can be a complete gene or small sequence

 Denaturing is the first step in PCR, in which the DNA strands are separated by heating to 95°C.

 Primers range from 15 to 30 nucleotides, are single-stranded, and are used for the complementary building blocks of the target sequence.  A primer for each target sequence on the end of your DNA is needed. This allows both strands to be copied simultaneously in both directions.

 Forward primer TTAACGGCCTTAA... TTTAAACCGGTT AATTGCCGGAATT >  Reverse primer < AAATTTGGCCAA TTAACGGCCTTAA... TTTAAACCGGTT  The primers are added in excess so they will bind to the target DNA instead of the two strands binding back to each other

 Annealing is the process of allowing two sequences of DNA to form hydrogen bonds. The annealing of the target sequences and primers is done by cooling the DNA to 55°C.

 Taq stands for Thermus aquaticus, which is a microbe found in 176°F hot springs in Yellow Stone National Forest.  Taq produces an enzyme called DNA polymerase, that amplifies the DNA from the primers by the polymerase chain reaction, in the presence of Mg.

 Denaturalization: 94°- 95°C  Primer Annealing: 55°- 65°C  Extension of DNA: 72°  Number of Cycles: 25-40

 10x assay buffer  2.5mM dNTP mix  Template DNA()  Forward primer  Reverse primer  Taq DNA pol  Sterile water  1%Agarose gel  Reference marker  Bromophenol blue dye

Setting up PCR 1. Add the following reagents to the PCR tube in the following order:  10x assay buffer: 05µl  2.5mM dNTP mix: 03µl  Template DNA(): 05 µl  Forward primer: 01µl  Reverse primer: 01µl  Taq DNA pol: 01µl  Sterile water: 34µl  Total reaction volume:50µl

2. Mix the contents gently and put the tube in the thermo cycler PCR Amplification 3.Carry out the amplification in a thermo cycler for 30 cycles using following reaction conditions: Pre denaturation : 94°c for 1min Denaturation : 94°c for 30 sec Annealing : 56 °c for 30 sec Extension : 72°c for 1 min Final Extension : 72°c for 10min

4. Prepare 1% agarose gel 5. Load 2µl of the ready to use marker provided and also load the amplified DNA sample. 6. Run the samples at 100volts for 1-2 hours till the tracking of the length of the gel. Dye reaches 3/4 th. 7. Visualize the gel under UV transilluminator