MOLECULAR TECHNIQUES
Polymerase Chain Reaction (PCR)
Purpose: Selectively amplify a specific region of DNA from a trace sample
Components: DNA template (containing nucleotide sequence of interest), DNA primers (specific to
and flank the sequence of interest), dNTPs (in excess), thermostable DNA polymerase (e.g. Taq
polymerase - enzyme is stable at high temperatures and is not denatured by repeated heat
treatments), PCR reaction buffer
Equipment: Thermal cycler
Practical Steps:
1) Denaturation of DNA template
The reaction mixture is heated to 95oC for 30 s to denature the DNA template (separate it into
single-stranded DNA) by breaking the hydrogen bonds between the two strands
2) Annealing of primers
The reaction mixture is cooled to 54oC for 1 min (in the presence of large excess of the two
sets of DNA primers) to allow the primers to anneal specifically to complementary sequences
at the 3’ end of single-stranded DNA templates via hydrogen bonds (hybridisation)
3) Extension of primers
The reaction mixture is heated to 72oC for 2 min to allow for DNA synthesis via extension of
primers in 5’ to 3’ direction catalysed by thermostable Taq DNA polymerase
Advantages Limitations
● Extremely sensitive - can amplify ● Extremely sensitive - risk of
sequences from minute amounts of DNA contamination resulting in amplification
of non-target sequences
● Rapid and can be easily automated
● Errors during DNA polymerisation - Taq
● Robust; permits amplification of specific DNA polymerase lacks 3’ to 5’
sequences from material in which DNA is exonuclease activity
badly degraded or difficult to isolate
● Can only efficiently amplify DNA up to a
few thousand base-pairs
● Prior sequence information is needed for
construction of primers to ensure
specificity
● Can only amplify nucleic acids (not
proteins)
Copyright © 2019 tonyndr
Polymerase Chain Reaction (PCR)
Purpose: Selectively amplify a specific region of DNA from a trace sample
Components: DNA template (containing nucleotide sequence of interest), DNA primers (specific to
and flank the sequence of interest), dNTPs (in excess), thermostable DNA polymerase (e.g. Taq
polymerase - enzyme is stable at high temperatures and is not denatured by repeated heat
treatments), PCR reaction buffer
Equipment: Thermal cycler
Practical Steps:
1) Denaturation of DNA template
The reaction mixture is heated to 95oC for 30 s to denature the DNA template (separate it into
single-stranded DNA) by breaking the hydrogen bonds between the two strands
2) Annealing of primers
The reaction mixture is cooled to 54oC for 1 min (in the presence of large excess of the two
sets of DNA primers) to allow the primers to anneal specifically to complementary sequences
at the 3’ end of single-stranded DNA templates via hydrogen bonds (hybridisation)
3) Extension of primers
The reaction mixture is heated to 72oC for 2 min to allow for DNA synthesis via extension of
primers in 5’ to 3’ direction catalysed by thermostable Taq DNA polymerase
Advantages Limitations
● Extremely sensitive - can amplify ● Extremely sensitive - risk of
sequences from minute amounts of DNA contamination resulting in amplification
of non-target sequences
● Rapid and can be easily automated
● Errors during DNA polymerisation - Taq
● Robust; permits amplification of specific DNA polymerase lacks 3’ to 5’
sequences from material in which DNA is exonuclease activity
badly degraded or difficult to isolate
● Can only efficiently amplify DNA up to a
few thousand base-pairs
● Prior sequence information is needed for
construction of primers to ensure
specificity
● Can only amplify nucleic acids (not
proteins)
Copyright © 2019 tonyndr
