To perform amplification of specific target sequence from genomic DNA using polymerase chain reaction.

PRINCIPLE :

Polymerase chain reaction is an invitro method of enzymatic synthesis of specific DNA sequence developed by Kary Mullis in 1983. It is a very simple and inexpensive technique for characterizing, analyzing and synthesizing any specific piece of DNA (or) RNA from virtually any living organisms - plant/animal/virus/bacteria. It exploits the natural function of the polymerase present in all living things to copy genetic material and perform polymerase chain reaction consists of three basic steps.

1. Denaturation

2. Annealing

3. Extension

1. DENATURATION :

During this step, the two strands melt open to form single stranded DNA and all enzymatic reaction step is generally carried out at 92-96 degree celcius.

2. ANNEALING :

Annealing of primers to each original strands for new strands synthesis is carried out between 45-55 degree celcius.

3. EXTENSION :

The polymerase adds dNTPs complementary to the template at the 3' ends of the primers. Since both strands are copied during polymerase chain reaction, there is an exponential increase in the number of copies of the genes.

FACTORS AFFECTING AMPLIFICATION :

1. REACTION COMPONENTS :

a). SAMPLE VOLUME :

Most amplifications are performed at 20 micro litre (or) 50 microlitre (or) 100 microlitre in 0.2 ml (or) 0.5ml centrifuge tubes.

Larger volumes donot allow adequate thermal equilibrium of the reaction mixture.

b). TEMPLATE DNA :

Generally nanogram amount of plasmid DNA (or) microgram amount of genomic DNA is used for PCR.

Higher amounts of template DNA inhibits (or) results in non-specific amplification.

c) PRIMERS :

Polymerase chain reaction needs two primers, a forward and a reverse primer. Primers are synthetic oligonucleotides usually ranging from 15-30 bases.

Primers are designed such that at the 3' ends, they donot have more than two bases complementary to each other, as this results in primer-dimer formation.

G+C content is in the range of 40-60% . The melting temperature of both forward and reverse primers is usually the same.

Low concentration of primers result in poor yield of the specific product and high concentraion of primers is between 0.1-1 micro meter.

d). DEOXYNUCLEOTIDE - TRIPHOSPHATE :

The final concentration of each dNTP [dATP, dGTP, dCTP, dTTP] in a standard amplification reaction is 200 micro metre.

It is important to keep the dNTP concentration above the estimated Km of each dNTP(10-15micro metre) for best base incorporation.

E). TAQ DNA POLYMERASE BUFFER :

The 10x assay buffer contains 100 mM Tris Hcl (H-9) 500 mM potassium chloride, 15mM magnesium chloride and 0.1% w/v gelatin.

Mg2+ is an essential cofactor required for the activity of TAQ DNA polymerase. Low concentration of magnesium will result in no amplification and high amounts may lead to the production of unwanted products.

F) TAQ DNA POLYMERASE :

TAQ DNA polymerase is a 94 KD, thermostable DNA polymerase. Optimal temperature for TAQ DNA polymerase activity is 72 degree celcius.

It lacks 3' ->5' exonuclease activity but has 5'->3' exonuclease and 5'->3' polymerase activity. For most of the amplification reaction, 1.5 to 2u of enzyme is recomended as higher enzyme concentration leads to non-specific amplification.

2. TEMPERATURE CYCLING :

Temperature cycling parameters depending greatly on the template, primers and amplification apparatus.

INITIAL DENATURATION STEP :

Complete denaturation of template DNA at the start of the PCR reaction is of key importance. In complete denaturation results in inefficient utilization of template in the first amplification cycle and is a pooe yield of PCR products. It is generally performed at 95 degree celcius for 1-3 minutes, depending on the GC content of the template. If longer initial denaturation (or) temperature id necessary, then TAQ DNA polymerase can be added after this temperature may affect the enzyme stability. Initial denaturation step is performed only once at the beginning of the reaction.

b). DENATURATION :

Subsequent denaturation steps are performed for a short time of 30 seconds to 1 minute at 94 degree celcius for 30 cycles. This is sufficient,  since the PCR product synthesized in the first amplification cycle is significantly shorter than the template DNA and completely denatures under these conditions. Certain additives like Dimethyl Sulfonide/glycerol/formamide are used to facilitate DNA denaturation depending on the GC content.

c) ANNEALING :

The optimal annealing temperature is generally 5 degree celcius lower than the melting temperature of primer- template DNA duplex, performed for 30 seconds to 1 minute. Non-specific products are obtained in addition to the expected product, the annealing temperature is optimized by increasing it stepwise by 1-2 degree celcius.

d) EXTENSION :

Primer extension, resulting in synthesis of new DNA strand is carried out at 72 degree which is optimal temperature for TAQ DNA polymerase activity. The amplification  time is determined by length of the sequence to be amplified. For every 1 KB larger DNA, one minute time is recommended. Number of PCR cycle depends on the amount of template DNA in the reaction mix and on the expected yield of the product.

e). FINAL EXTENSION :

As DNA synthesis proceeds, it becomes less efficient as most of the components get used up. Hence following the last cycle enzyme is allowed to finish any incomplete synthesis by carrying out a final extension at 72 degree celcius for 5-15 minutes.

MATERIALS REQUIRED :

1. 10x assay buffer

2. Nuclease free-water

3. 10mM dNTP mix

4. Template DNA

5. PCR tubes(0.5ml)

6. 100bp ladder

7. TAQ DNA polymerase

8. 50x TAE buffer

9. Forward primer, Reverse primer

10. Agarose

11. Mineral oil

12. Gel ladding dye

13. 25x gel loading buffer

14. Etidium bromide

15. Double distilled water

16. Ice bucket

17. Gloves

18. Micropipette

19. Thermal Cycler

20. UV-transilluminator

PROCEDURE :

SETTING UP PCR :

1. Add the following reagents to the PCR tube in the following order.

sterile water - 38 micro litre

10x assay buffer - 5 micro litre

10 mM dNTP mix - 3ml

Template DNA (100 ng/micro litre) = 1micro litre

Forward primer (100ng/micro litre) =1 micro litre

Reverse primer (100 ng/micro litre) = 1 micro litre

TAQ DNA polymerase(3 unit/ micro litre) = 1micro litre

Total reaction volume = 50 micro litre

Mix the contents gently and layer the reaction mix with 50 micro litre of mineral oil, to prevent evapouration.

3. PCR AMPLIFICATION :

Carryout the amplification in a thermocycler for 30 cycles using the following reaction conditions :

Initial Denaturation ---94 C[for 1 minute]

Denaturation         ---94 C[for 30 second]

Annealing             ---48[for 30 seconds]

Extension             ---72 C[for 60 seconds]

Final Extension    ----72 C[for 2 minutes]

4. The amplified DNA is mixed with 5 micro litre of gel loading buffer.

5. COst 1 % agarose gel, load the samples into the well and run the gel for two hours.

Examine the gel on a UV transilluminator to visualize the DNA bands.

RESULT :

The PCR amplified sample is electrophoresed along with the markerr provided and visualized under the UV-transilluminator.