The Single Base Extension Reaction

Reaction protocols suitable for ThemtoSequenase and AmpUTaq FS DNA polymerases have been previously described in detail [9] tbe protocols are similar for rTth or Tth DNA polymerase. Reaction mixtures typically included 0.125 units/pl of Tth DNA polymerase, 0.5 pM of each genotyping primer, and 0.25 to 0.50 volumes of polished PCR product per volume of final reaction mixture. A typical reaction volume is 10 pi. 

The recommended thermal cycling protocol (1 second 94° C - 2.5 minutes 37° C, for 25 thermal cycles, followed by a 4° C hold) does not require a third, higher-temperature primer extension step as commonly used in PCR. This low-temperature extension permits efficient extension of short and A+T rich primers. Thermal cycling 25 times provides a linear amplification of primer extension product, which may exceed the concentration of the PCR target sequence up to about 25-fold. 

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Incubated the mixture at 37°C for 45 min to degrade the excess PCR primers and excess dNTP. Heat inactivate the enzymes were at 80°C for 15 min before the single-base extension reaction. 

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