Polymerase Chain Reaction (PCR) cont.

Polymerase Chain Reaction (PCR) cont.

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

• Properties of DNA polymerases used in PCR

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_ms and T_mp calculations (0.05 M K⁺)