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PCRPolymerase Chain

Reaction

Metode enzimatis untuk melipatgandakan secara eksponensial suatu sekuen nukleotida tertentu

It was invented in 1983 by Dr. Kary Mullis, for which he received the Nobel Prize in Chemistry in 1993.

PCR

• Metode yg mengamplifikasi atau mengkopi sejumlah kecil rangkaian DNA atau satu molekul DNA.

• Memisahkan gen-gen berkopi tunggal dri sekelompok sekuen genom.

Why “Polymerase”?

It is called “polymerase” because the only enzyme used in this reaction is DNA polymerase.

Why “Chain”?

It is called “chain” because the products of the first reaction become substrates of the following one, and so on.

The Reaction

THERMOCYCLERPCR tube

PCR Machine / Thermocycler

• Components of PCR– Template DNA– dNTPs (dATP, dTTP, dCTP & dGTP)– primers– Taq DNA polymerase– MgCl2

– PCR buffer

DNA template The DNA which will be amplified by the PCR

provide both the energy and nucleosides for the synthesis of DNA. It is important to add equal amounts of each nucleotide (dATP, dTTP, dCTP, dGTP) to the mixture to prevent mismatches of bases.

dNTP(deoxynucleotide triphosphate)

The role of Mg++ in PCR forms complexes with dNTPs that are the actual substrates for Taq Polymerase. When Mg++ is too low, primers fail to an-neal to the target DNA. When Mg++ is too high, the base pairing becomes too strong and the amplicon fails to denature completely when you heat to 94°C.

promotes specific annealing of primers to the PCR template. K+ binds to phosphate groups on double-stranded DNA, stabilizing primer annealing.

MgCl2

KCl

Short pieces of DNA (20-30 bases) that bind to the DNA template allowing Taq DNA polymerase enzyme to initi-ate incorporation of the deoxynucleotides. Both specific and universal primers can be used.

Primers

A heat stable enzyme that adds the deoxynucleotides to the DNA template.

Taq DNA poly-merase

keeps the mixture at the proper pH so the PCR reaction will take place.

Buffer

• Three major phases in PCR: – Denaturing (94ºC)– Annealing (55ºC)– Extension (72ºC)

• The total time to perform a standard PCR is approximately 4 hours.

DenaturationAt temperatures above 90°C, double-stranded DNA denatures or "melts". That means the weak hydrogen bonds that usually hold the two complementary strands together at normal temperatures are disrupted resulting in two single stranded DNA strands

PCR cycling program

DNA melting

The three parts of the polymerase chain reaction are carried out in the same vial, but at different temperatures. The first part of the process separates the two DNA chains in the double helix. This is done simply by heating the vial to 95 oC for 30 seconds.

double-stranded DNA single-stranded DNA

After separating the DNA strands, the temperature is lowered so the primers can attach themselves to the single DNA strands. The temperature of this stage depends on the primers and is usually 5°C below their melting temperature (45-60°C).

Annealing

Primer annealing

The primers cannot bind to the DNA strands at such a high temperature, so the vial is cooled to 55 oC. At this temperature, the primers bind or "anneal" to the appropriate location in the DNA strands. This takes about 20 seconds.

single-stranded DNA + primer annealed DNA

The final step of the reaction is to make a complete copy of the templates.

Primers are short, artificial DNA strands — often not more than 50 and usually only 18 to 25 base pairs long — that are com-plementary to the beginning or the end of the DNA fragment to be amplified

Primers

GAGGTAACCACACCAGA → 4Gs, 5Cs, 7As, 1T Tm = {4×(4+5)}+{2×(7+1)} = (4×9)+(2×8) = 36+16 = 52 Tm = 52℃ ∴ ℃

Estimation of the melting and annealing temperatures of primer:If the primer is shorter than 25 nucleotides, the approx. melt-ing temperature (Tm)Tm=4(G+C) + 2 (A+T)

Annealing temperature should be approx. 5 lower than the Tm

Examples of bacteria universal primer sequences are:Forward 5' GAT CCT GGC TCA GGA TGA AC 3' (20 mer)Reverse 5' GGA CTA CCA GGG TAT CTA ATC 3' (21 mer)

Finally, the DNA polymerase has to copy the DNA strands. It starts at the annealed primer and works its way along the DNA strand. The extension temperature depends on the DNA polymerase.

Taq polymerase extends optimally at a temperature of 72°C. The time for this step depends both on the DNA polymerase itself and on the length of the DNA fragment to be amplified

Extension

Primer extensionThe Taq polymerase begins adding nucleotides to the primer and eventually makes a complementary copy of the section of the template that lies between the primers. This completes one PCR cycle.

primer-annealed DNA primer-extended DNAwith Taq polymerase anddATP, dTTP, dGTP, dCTP

(1) 90ºC, open the DNA double-stranded helix.

(2) 50ºC, hybridization with primer.

(3)75ºC, DNA synthesize.

Repeating for 20~30 times. The amount doubles in every cycle.

PCR AnimationPlease click here.

1st cycle 2nd cycle 3rd cycle

Process

Denature

Anneal Primer

Replicate DNA

Extraction of DNA

Add proteaseAdd lysis buffer

Vortex each tube for 15 sec. to ensure proper mixing.

Incubate each tube for 10 min. at 56°C.

Centrifuge each to remove any mixture that may be on the lid.

Add 210µl of ethanol,vortex and then centrifuge again.

Collection DNA

The plate is then placed in the automated reader,where each well is read spectrophotometrically.

Preparation the master mixMaster Mix•10x Buffer - 10 µl•MgCl2 - 6 µl•dNTP mix - 0.8 µl of each nucleotide•F5F primer - 2 µl•F5R primer - 2 µl •Taq polymerase - 0.5 µl•Sterile H2O - 73.7 µl

Place 5µl of sample and 95µl of master mix in vials and place these vials in a PCR panel, whichwill then be placed in the thermocycler for theDNA amplification cycles.

Designing PCR programs

APLIKASI PCR

- Deteksi dini penyakit infeksi dan herediter- Test Paternity & Maternity - Uji GMO (genetically modified organism)- Uji keseragaman/kemurnian genetis- Test forensik- Peningkatan kualitas & kuantitas produk biologis- Pembuatan produk rekombinan (vaksin, hormon, dll.)

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