Factors affecting Polymerase Chain

Reaction and its Optimization

PRESENTED BY :Smruti Ranjan Dixit

Adm no- 10PBG/13

6th yr Msc(Ag)

DEPT OF PBG

Contents

• What is PCR?• Basic requirements for PCR reactions• Steps in PCR• Instrumentation• Factors Affecting PCR• Elaboration of Factors• PCR Optimization

What is PCR?

• PCR is a technique that takes specific

sequence of DNA of small amount and

amplifies it to be used for further testing.• In vitro technique• To amplify a lot of double-stranded

DNA molecules (fragments) with same (identical) size and sequence by enzymatic method and cycling condition.

Basic requirements for PCR reaction

• 1) DNA sequence of target region must be known.

2) Primers - typically 20-30 bases in size.These can be readily produced by commercial companies. Can also be prepared using a DNA synthesizer

Basic requirements for PCR reaction

• 3) Thermo-stable DNA polymerase - eg Taq polymerase which is not inactivated by heating to 95C

4) DNA thermal cycler - machine which can be programmed to carry out heating and cooling of samples over a number of cycles.

Steps In PCR

• 1. Denaturation of ds DNA template

• 2. Annealing of primers

• 3. Extension of DNA molecules

Denaturation

• Temperature: 92-94 degree C• Time is 2 min.• Double stranded DNA melts single stranded

DNA

92C

3’5’

3’ 5’

+

5’3’

5’ 3’

Annealing

• Temperature: ~50-70C (dependant on the melting temperature of the expected duplex)

• Primers bind to their complementary sequences

5’3’

5’ 3’

Forward primer Reverse primer

Extension

• Temperature: ~72C• Time: 0.5-3min• DNA polymerase binds to the annealed primers and

extends DNA at the 3’ end of the chain

Taq

5’3’

Taq5’

Cycling

Products of Extension

3’5’

3’ 5’

3’5’

3’ 5’

Taq

Taq

Instrumentation

Factors Affecting PCR

Primer Length Base Composition Length And Sequence of Target DNA Primer Sequence Denaturing temprature and time Annealing temprature and time Melting Temprature Extension Temprature and time Conc. Of different reagents No of Cycles

Primer Length

• Efficiency of PCR is affected by Primer Length.• It declines if the primers used for PCR are too

long.• The Longer the Primer the higher annealing

temprature required.• Generally speaking, the length of primer has

to be at least 15 bases to ensure uniqueness. Usually, we pick primers of 17-28 bases long.

Base Composition

• Base composition affects hybridization specificity,melting/annealing temperature and internal stability.

• We shall avoid long(A+T) and (G+C) rich region if possible.

• Usually, average (G+C) content around 50-60% will give us the right melting/annealing temperature for ordinary PCR reactions, and will give appropriate hybridization stability.

Length and Sequence Of Target DNA

• The Efficiency of Amplification declines with an increase in length of the target sequence .

• The GC rich sequence in target DNA may form secondary structures in the single strands produced by Denaturation that could reduce PCR efficiency.

Primer Sequence

• Primer sequence also affects PCR efficiency.• PCR primers must not have

selfcomplementary regions as this would lead to hairpin formation with in primer molecules and make then unsuitable for PCR .

• In addition 2 primers used in a PCR must not have regions complementary to each other because this would result in primer dimer formation.

Denaturing Temprature and Time

• Normally the denaturation time is 2 min at 94oC .

• it is possible, for short template sequences, to reduce this to 30 sec or less.

• Increase in denaturation temperature and decrease in time may also work .

Annealing Temprature and Time

• If Annealing temprature is too high, pairing between the primers and template DNA will not take place and the PCR will fail.

• An ideal annealing temprature must be low enough to enable hybridization between primer and template, but high enough to prevent amplification of nontarget sites.

• Ideal Annealing temp is 55-60 degree c and time is 1 min.

Melting Temprature

• Tm is the temp at which the 2 strands of the duplex dissociate.

• Tm = 4(G + C) + 2(A + T)oC.

Where G+C is the no of G and C nucleotides

A+T is no of A and T nucleotides in the Primer sequence.

• Annealing temp is 1-2 degree c below the value of melting temprature.

Extension Temprature and Time

• This is normally 70 – 72OC, for 0.5 - 3 min. • At around 70oC the activity is optimal, and

primer extension occurs at up to 100 bases/sec.

• About 1 min is sufficient for reliable amplification of 2kb sequences (Innis and Gelfand, 1990).

• Longer products require longer times: 3 min is a good bet for 3kb and longer products.

Conc of Different Reagents

• Magnesium chloride: .5-2.5mM

• Buffer: pH 8.3-8.8

• dNTPs: 20-200µM

• Primers: 0.1-0.5µM

• DNA Polymerase: 1-2.5 units

• Target DNA: ≤ 1 µg

No of Cycles

• The number of amplification cycles necessary to produce a band visible on a gel depends largely on the starting concentration of the target DNA:

• Innis and Gelfand (1990) recommend from 40 - 45 cycles to amplify 50 target molecules, and 25 - 30 to amplify 3x105 molecules to the same concentration.

PCR OPTIMIZATION

• Primer Length: 17-28 bases.This range varies.• Base composition: average (G+C) content

around 50-60%;avoid long (A+T) and (G+C) rich region if possible.

• Optimize base pairing: it’s critical that the stability at 5’ end be high and the stability at 3’ end be relatively low to minimize false priming.

PCR OPTIMIZATION

• Denaturation Temp & Time :Normally the denaturation time is 2 min at 94oC .

• Annealing Temp & Time : 40-60 degree c for 1 min.

• Extension Temp : 70-72 degee C for 2 min .• Conc of diff reagents :Magnesium chloride: .5-

2.5mM,Buffer: pH 8.3-8.8,dNTPs: 20-200µM,Primers: 0.1-0.5µM,DNA Polymerase: 1-2.5 units,Target DNA: ≤ 1 µg

PCR OPTIMIZATION

• Minimize internal secondary structure: hairpins and dimers shall be avoided. primer self-complementarity (ability to form 2o structures such as hairpins) should be avoided.

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