| 摘要 |
<p>A method for improving the priming specificity of an oligonucleotide primer that is non-unique in a nucleic acid template includes selecting a continuous stretch of several nucleotides in the template DNA such that at least one of the four bases does not occur in said stretch; bringing the template DNA in contact with a non-unique primer partially or fully complementary to the sequence immediately upstream of the selected sequence stretch; carrying out a polymerase-mediated differential extension of the primer in the presence of a subset of deoxyribonucleotide triphosphates that does not contain the base complementary to the base absent in the selected sequence stretch at a temperature sufficiently low for allowing for the extension of the non-unique primer; and carrying out polymerase-mediated extension reactions in the presence of all four natural deoxyribonucleotide triphosphates or modifications thereof at a high temperature at which discrimination occurs against priming sites of the non-unique primer where the differential extension has not made the primer sufficiently stable to prime at the high temperature, and where the primer extended at the selected stretch is sufficiently stable to prime.</p> |
