PCR and Southern Hybridisation
Polymerase chain reaction
What it does:
Requires a minimum of sequence information on DNA molecule of
interest
Gives the exponential amplification of a single gene
Enables identification, amplification & isolation of DNA of
interest from a minute amount of a mixed DNA population
Thermus aquaticus – Taq DNA polymerase: PCR depends on the use
of this particular DNA polymerase isolated from this bacterium which lives
in hot springs. Its enzymes (including DNA polymerase) are not only
tolerant to high temperatures but will work normally at them.
Taq can withstand the high temperatures (95 degrees) required to
denature double stranded DNA – it works best at high temperatures
(75 degrees)
Taq requires:
- Single stranded DNA template
- Short DNA primer base-paired to template
- Supply of dNTPs
PCR is carried out in a computer- controlled heating block that can
be programmed to cycle through precise changes in temperature for
specific time periods
How it works:
Mix:
- DNA
- Forward & reverse primers (synthetic oligonucleotide primers are
synthesised complementary to the 2 ends of the DNA molecule of
interest)
- Taq polymerase
- Buffer
- Water
Heat (94 degrees) to denature DNA
Cool (55 degrees) so that primers anneal (base pair/ hybridise) to
complementary sequences
Heat (74 degrees) to synthesise first “long products” – DNA
strands go beyond the primer on the other strand
Repeat cycles 30-40 times
“Short products” accumulate exponentially – DNA strands do not
go beyond the primer on the other strand
, PCR: amplification:
30 cycles of amplification gives 228 fragments
During each cycle there is a doubling of the amount of DNA
synthesised, until there is enough to see on a normal agarose gel or
to clone
Very sensitive procedure:
- Can start with 1 molecule of DNA
- Risk of contamination
- Primers must be highly specific to the gene of interest
- PCT often needs optimisation
Primer design:
Primers are usually 17-22 bases in length
Usually end in Gs & Cs – 3 hydrogen bonds, bind tighter than A or T
Length:
Short primers (e.g. 8-mers) will hybridise at random due to chance
(will randomly stick to other parts of the genome)
Longer primers (e.g. 17-mers) have a high probability of being
specific to the gene/ region of interest
Sequence:
C & G have 3 H bonds
A & T have 2 H bonds
These bonds affect the stability of base-pairing
- Tm – melting temperature at which the correct base-paired hybrid
dissociates
- Can be calculated empirically: Tm = (4 x [G + C]) + (2 x [A +T]) oC
Tm controls annealing temperature of primers to template – should
be similar for both primers
Polymerase chain reaction
What it does:
Requires a minimum of sequence information on DNA molecule of
interest
Gives the exponential amplification of a single gene
Enables identification, amplification & isolation of DNA of
interest from a minute amount of a mixed DNA population
Thermus aquaticus – Taq DNA polymerase: PCR depends on the use
of this particular DNA polymerase isolated from this bacterium which lives
in hot springs. Its enzymes (including DNA polymerase) are not only
tolerant to high temperatures but will work normally at them.
Taq can withstand the high temperatures (95 degrees) required to
denature double stranded DNA – it works best at high temperatures
(75 degrees)
Taq requires:
- Single stranded DNA template
- Short DNA primer base-paired to template
- Supply of dNTPs
PCR is carried out in a computer- controlled heating block that can
be programmed to cycle through precise changes in temperature for
specific time periods
How it works:
Mix:
- DNA
- Forward & reverse primers (synthetic oligonucleotide primers are
synthesised complementary to the 2 ends of the DNA molecule of
interest)
- Taq polymerase
- Buffer
- Water
Heat (94 degrees) to denature DNA
Cool (55 degrees) so that primers anneal (base pair/ hybridise) to
complementary sequences
Heat (74 degrees) to synthesise first “long products” – DNA
strands go beyond the primer on the other strand
Repeat cycles 30-40 times
“Short products” accumulate exponentially – DNA strands do not
go beyond the primer on the other strand
, PCR: amplification:
30 cycles of amplification gives 228 fragments
During each cycle there is a doubling of the amount of DNA
synthesised, until there is enough to see on a normal agarose gel or
to clone
Very sensitive procedure:
- Can start with 1 molecule of DNA
- Risk of contamination
- Primers must be highly specific to the gene of interest
- PCT often needs optimisation
Primer design:
Primers are usually 17-22 bases in length
Usually end in Gs & Cs – 3 hydrogen bonds, bind tighter than A or T
Length:
Short primers (e.g. 8-mers) will hybridise at random due to chance
(will randomly stick to other parts of the genome)
Longer primers (e.g. 17-mers) have a high probability of being
specific to the gene/ region of interest
Sequence:
C & G have 3 H bonds
A & T have 2 H bonds
These bonds affect the stability of base-pairing
- Tm – melting temperature at which the correct base-paired hybrid
dissociates
- Can be calculated empirically: Tm = (4 x [G + C]) + (2 x [A +T]) oC
Tm controls annealing temperature of primers to template – should
be similar for both primers
