Selective amplification of desired nucleic acid regions in a target sequence

A method for selective amplification of desired amplicons in a two-stage polymerase chain reaction, comprising the steps of:

• providing a target genetic material;
• denaturing the target genetic material;
• adding a set of target primers under conditions allowing annealing of primers to the target genetic material;
• each primer in said set of target primers comprising
• a target specific sequence,
• a mismatch control sequence, and
• a generic hybridization sequence;
• said generic hybridization sequence being identical in each target primer;
• said mismatch control sequence and generic hybridization sequence being in close proximity to each other;
• and
• said set of target primers comprising at least two primer pairs adapted for amplification of at least two overlapping desired amplicons in the target genetic material, such that there is a possibility of obtaining at least one undesired amplicon in the region of overlap;
• said set of target primers being further characterized in that any pair of primers that flank a desired amplicon sequence have non-matching mismatch control sequences, while any pair of primers that flank an undesired amplicon sequence have matching mismatch control sequences;
• carrying out a first multiplex polymerase chain reaction resulting in overlapping amplicons corresponding to different possible pair-wise combinations of primers in the first set of primers, said overlapping amplicons being maintained under conditions such that they self-hybridize through base-pair coupling between the complementary sequences resulting from the generic hybridization sequence in each primer;
• treating said overlapping amplicons under selection conditions that are such that desired amplicons denature because of non-complementary sequences resulting from the non-matching mismatch control sequences, while undesired amplicons stay self-hybridized because of additional complementary sequences resulting from the matching mismatch control sequences;
• adding generic primers, each comprising a sequence which is complementary to the generic hybridization sequence;
• carrying out a second polymerase chain reaction, resulting in the selective amplification of desired amplicons.