Products nucleotide misincorporation

Retroviral RTases are multifunctional enzymes that exhibit at least three distinct enzymatic activities (i) RNA-directed DNA polymerase, (ii) DNA-directed DNA polymerase in the conversion of ssDNA to dsDNA, and (iii) RNase H that selectively removes the RNA moiety from a RNA DNA heteroduplex. Reverse transcriptases lack both 3 5 - and 5 —> 3 -exonuclease activities, and exhibit relatively high frequencies of nucleotide misincorporation. The RNA- and DNA-dependent polymerase activities are physically inseparable. Reverse transcriptases from avian retroviruses, but not from mammalian retroviruses, exhibit a fourth activity, DNA endonuclease, that is essential for the integration of the dsDNA intermediate into the host chromosome and for the productive infection of the retrovirus. 

Mutagenic PGR. More recently, methods have been developed to use the PGR reaction to randomly mutagenize a defined sequence (25). The Taq polymerase used in PGR misincorporates nucleotides in a random fashion if manganese dichloride [7773-01 -5] MnGl2, is included in the reaction buffer during PGR. The Hbrary of mutagenized PGR products can be screened for the desired phenotype. 

For most applications the error rate of the polymerase has no effect on the quality of the sequence information obtained. This is because the amplified product (sequencing template) is a population of molecules with only a relatively small fraction of the copies containing misincorporated nucleotides at any one position (Table II). The fraction of template molecules that will contain a misincorporated base is dependent on the starting number of target molecules and the cycle in which the error occurred. The fraction is very small, undetectable in most applications, if the number of starting molecules is at least 10. 

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