Factors affecting PCR

1. Factors affecting
Polymerase Chain
Reaction and its
Optimization
PRESENTED BY :
Smruti Ranjan Dixit
Adm no- 10PBG/13
6th
yr Msc(Ag)
DEPT OF PBG

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

3. 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.

4. 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

5. 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.

6. Steps In PCR
• 1. Denaturation of ds DNA template
• 2. Annealing of primers
• 3. Extension of DNA molecules

7. 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’

8. 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

9. 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’

10. Cycling

11. Products of Extension
3’5’
3’ 5’
3’5’
3’ 5’
Taq
Taq

12. Instrumentation

13. 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

14. 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.

15. 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.

16. 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.

17. 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.

18. Denaturing Temprature and Time
• Normally the denaturation time is 2 min at
94o
C .
• 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 .

19. 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.

20. 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.

21. Extension Temprature and Time
• This is normally 70 – 72O
C, 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.

22. 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

23. 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.

24. 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.

25. PCR OPTIMIZATION
• Denaturation Temp & Time :Normally the
denaturation time is 2 min at 94o
C .
• 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

26. 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.

27. THANK YOU