Polymerase Chain Reaction (PCR) cont.

 

Polymerase Chain Reaction (PCR) cont.

 

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Choice of Polymerases for PCR

• One of the important advances which allowed development of PCR was the availability of thermostable polymerases.
• This allowed initially added enzyme to survive temperature cycles approaching 100 °C.

Thermostable DNA polymerases and their sources

DNA Polymerase	Natural or recombinant	Source
Taq	Natural	Thermus aquaticus
Amplitaq®	Recombinant	T. aquaticus
Amplitaq (Stoffel fragment)®	Recombinant	T. aquaticus
Hot Tub™	Natural	Thermus flavis
Pyrostase™	Natural	T. flavis
Vent™	Recombinant	Thermococcus litoralis
Deep Vent™	Recombinant	Pyrococcus GB-D
Tth	Recombinant	Thermus thermophilus
Pfu	Natural	Pyrococcus furiosus
ULTma™	Recombinant	Thermotoga maritima

• Properties of DNA polymerases used in PCR

	Taq /Amplitaq®	Stoffel fragment	Vent™	Deep Vent™	Pfu	Tth	ULTma™
95 °C half-life	40 min	80 min	400 min	1380 min	>120 min	20 min	>50 min
5'3' exo	+					+
3'5' exo			+	+	+		+
Extension rate (nt/sec)	75	>50	>80	?	60	>33	?
RT activity	Weak	Weak	?	?	?	Yes	?
Resulting ends	3' A	3' A	>95% blunt	>95% blunt	blunt	3' A	blunt
Strand displacement			+	+
M.W. (kDa)	94	61	?	?	92	94	70

 

Buffers and MgCl₂ in PCR reactions

A typical reaction buffer for PCR would something like:

• 10 mM Tris, pH 8.3
• 50 mM KCl
• 1.5 mM MgCl₂
• 0.01% gelatin
• The MgCl₂ concentration in the final reaction mixture is usually between 0.5 to 5.0 mM, and the optimum concentration is determined empirically (typically between 1.0 - 1.5 mM). Mg²⁺ ions :
  • form a soluble complex with dNTP's which is essential for dNTP incorporation
  • stimulate polymerase activity
  • increase the Tm (melting temperature) of primer/template interaction (i.e. it serves to stabilize the duplex interaction

Generally,

• low Mg²⁺ leads to low yields (or no yield) and
• high Mg²⁺ leads to accumulation of nonspecific products (mispriming).

Primers

Primer design

• Generally, primers used are 20 - 30 mer in length. This provides for practical annealing temperatures (of the high temperature regimen where the thermostable polymerase is most active).
• Primers should avoid stretches of polybase sequences (e.g. poly dG) or repeating motifs - these can hybridize with inappropriate register on the template.
• Inverted repeat sequences should be avoided so as to prevent formation of secondary structure in the primer, which would prevent hybridization to template
• Sequences complementary to other primers used in the PCR should be avoid so as to prevent hybridization between primers (particularly important for the 3' end of the primer)
• If possible the 3' end of the primer should be rich in G, C bases to enhance annealing of the end which will be extended
• The distance between primers should be less than 10 Kb in length. Typically, substantial reduction in yield is observed when the primers extend from each other beyond ~3 Kb.

Melting temperature (Tm) of primers

• The Tm of primer hybridization can be calculated using various formulas. The most commonly used formula is:

(1) Tm = [(number of A+T residues) x 2 °C] + [(number of G+C residues) x 4 °C]

• This formula was determined originally from oligonucleotide hybridization assays, which were performed in 1 M NaCl, and appears to be accurate in lower salt conditions only for primers less than about 20 nucleotides in length.
• The common wisdom is that the Tm is more like 3-5 °C lower than the value calculated from this formula.

(2) Tms = 81.5 + 16.6(log10[J+]) + 0.41(%G+C) - (600/l)

• Where [J⁺ ] = the molar concentration of monovalent cations (e.g. Na⁺ from NaCl), and l = the length of oligonucleotide. (%G+C) is the actual percentage value and not a fractional representation (i.e. the value to insert for a primer which had 90 % G+C content would be "90" and not "0.90").
• This formula is reportedly useful for primers of 14 to 70 bases in length.

(3) Tmp = 22 + 1.46([2 x (G+C)] + (A+T))

• This formula is reportedly useful for primers of 20-35 bases in length.
• The calculated annealing temperature is only a reference temperature from which to initiate experiments.
  • The actual annealing temperature may be 3-12 °C higher than the calculated Tm.
  • The actual annealing temperature condition should be determined empirically.
  • The highest annealing temperature which gives the best PCR product should be used.
• Examples of T_m , T_m s and T_m p calculations (0.05 M K⁺ )

	G	A	T	C	Tm	Tm s	Tm p
15 mer	3	5	2	5	46	42	56
20 mer	6	5	4	5	62	52	67
30 mer	8	6	8	8	92	62	89

Calculating primer concentrations

• The molar concentration of a primer can be calculated based on the absorbance of the primer at 260 nm (A₂₆₀ ) and the molar extinction coefficient for the primer at this wavelength.
• The molar extinction coefficient for the primer can be calculated by knowing the sequence of the primer and then summing the molar extinction values for the individual bases which comprise the primer.
• The individual bases have the following molar extinction coefficients at 260 nm:
  • A 1.0 molar solution of dT has a value of 8,400 absorbance units at 260 nm.
  • A 1.0 molar solution of dA has a value of 15,200 absorbance units at 260 nm.
  • A 1.0 molar solution of dG has a value of 12,010 absorbance units at 260 nm.
  • A 1.0 molar solution of dC has a value of 7050 absorbance units at 260 nm.
• For example, the primer 5' TAGC 3' would have a molar extinction coefficient of 42,660 at 260 nm. Likewise, a 10 micromolar solution of this primer would give an absorbence of 0.427 at 260 nm.