Patent ID: 6207425
Filing Date: 2001-03-27
Classification: C12Q

Abstract:
A method for conducting a bi-directional PCR amplification of specific alleles which comprises subjecting DNA which may contain one or both of first and second alleles to a single one tube PCR under a thermal cycling condition utilizing an outer pair of primers P and Q and an inner pair of primers A and B, whereQ is complementary to the sense strand of both alleles in a region downstream of the sequence difference X which distinguishes the alleles and P is complementary to the anti-sense strand of both alleles in a region upstream of X;B has a region at its 3' end which is complementary to the sense strand of the first allele and A has a region at its 3' end which is complementary to the antisense strand of the second allele, each of A and B also has a non-complementary G+C-rich tail at its 5' end, and X occurs at or near the 3' end of each of A and B, whereby A and B are in two directions with partially overlapped 3' ends;whereby DNA which is heterozygous with respect to the two alleles results in simultaneous amplification of three overlapping sequences, PQ, PB and AQ, DNA which is homozygous with respect to the first allele results in simultaneous amplification of two overlapping sequences, PQ and PB, and DNA which is homozygous with respect to the second allele results in simultaneous amplification of two overlapping sequences, PQ and AQ, where PQ, PB and AQ, respectively, stand for the sequences extending from P to Q inclusive, from P to B inclusive, and from A to Q inclusive, wherein the method is conducted at a substantially constant annealing temperature in the range of about 50-65.degree. C. and with a ratio of concentrations of the outer and inner primers in the PCR mixture in the range of about 1:1 to 1:6.