Detection of differences in nucleic acids

Details Detection of differences in nucleic acids Detection of differences in nucleic acids

1996-12-20

2000-01-11

Marschel, Ardin H.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

536 2532, C12Q 168, C07H 2100

Patent

active

060134399

ABSTRACT:
A method is disclosed for detecting the presence of a difference between two related nucleic acid sequences. In the method a complex is formed comprising both strands of each sequence. Each member of at least one pair of non-complementary strands within the complex have labels. The association of the labels as part of the complex is determined as an indication of the presence of a difference between the two related sequences. The complex generally comprises a Holliday junction. In one aspect a medium suspected of containing said two related nucleic acid sequences is treated to provide partial duplexes having non-complementary tailed portions at one end. The double stranded portions of the partial duplexes are identical except for said difference. One of the strands of one of the partial duplexes is complementary to one of the strands of the other of the partial duplexes and the other of the strands of one of the partial duplexes is complementary to the other of the strands of the other of the partial duplexes. The medium is subjected to conditions that permit the binding of the tailed portions of the partial duplexes to each other. If there is a difference in the related nucleic acid sequences, a stable complex is formed comprising a Holliday junction. If no difference exists, the complex dissociates into duplexes. A determination is made whether the stable complex is formed, the presence thereof indicating the presence of the related nucleic acid sequences. The method has application in detecting the presence of a mutation in a target sequence or in detecting the target sequence itself.

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Mueller et al. T4 endonuclease VII cleaves the crossover strands of Holliday junction analogs. Proc. Natl. Acad. Sci. USA vol. 85, pp. 9441-9445, 1988.
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Birch; Nature; 381:445-446; Simplified Hot Start PCR; May 30, 1996.

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