Patent ID: 6258544
Filing Date: 2001-07-10
Classification: C12N,C12Q

Abstract:
A method of detecting whether a first double-stranded DNA has base differences as compared to a second double-stranded DNA, said method comprising:a) amplifying said first double-stranded DNA molecule using a pair of amplification primers to form an amplified first DNA wherein each strand of said amplified first DNA comprises a first region and a second region wherein said first region and said second region are reverse complements of each other;b) denaturing said amplified first DNA to form single strands of first DNA;c) allowing intrastrand-annealing of said single strands of first DNA wherein said first region on one strand of DNA anneals with said second region on said one strand of DNA to form intrastrand-annealed first DNA;d) extending said intrastrand-annealed first DNA to yield panhandle DNA which upon denaturation yields a single-stranded first DNA molecule comprising sequence of said both strands of double-stranded DNA;e) amplifying said second double-stranded DNA molecule using said pair of amplification primers from step (a) to form an amplified second DNA wherein each strand of said amplified second DNA comprises a first region and a second region wherein said first region and said second region are reverse complements of each other;f) denaturing said amplified second DNA to form single strands of second DNA;g) allowing intrastrand-annealing of said single strands of second DNA wherein said first region on one strand of DNA anneals with said second region on said one strand of DNA to form intrastrand-annealed second DNA;h) extending said intrastrand-annealed second DNA to yield panhandle DNA which upon denaturation yields a single-stranded second DNA molecule comprising sequence of said both strands of double-stranded DNA;i) electrophoresing said single-stranded first DNA and said second single-stranded DNA on a denaturing gradient gel;j) determining the migration distance of said single-stranded first DNA; andk) determining the migration distance of said single-stranded second DNA; wherein if said migration distance of step j) differs from said migration distance of step (k), then said first double-stranded DNA has base differences as compared to said second double-stranded DNA.