Detection of mismatches by resolvase cleavage on a solid support

Details Detection of mismatches by resolvase cleavage on a solid support Detection of mismatches by resolvase cleavage on a solid support

1997-06-24

1999-03-02

Zitomer, Stephanie W.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 912, 536 2433, 536 254, 935 77, 935 78, C12Q 168, C07H 2104, C12P 1934

Patent

active

058769412

ABSTRACT:
Disclosed is a method for detecting one or more mismatches in a test nucleic acid which preferentially hybridizes to a reference nucleic acid. The method involves a) providing the test nucleic acid in its single-stranded form; b) providing the reference nucleic acid immobilized on a solid support, the reference nucleic acid being in its single-stranded form and the solid support being compatible with a slot in an electrophoretic gel; c) contacting the single-stranded test nucleic acid with the single-stranded immobilized reference nucleic acid under conditions allowing heteroduplex formation, whereby the heteroduplex is immobilized on the solid support; d) contacting the immobilized heteroduplex with a resolvase (for example, T4 endonuclease VII) capable of recognizing at least one single base pair mismatch in the heteroduplex under conditions which permit the resolvase to cleave the heteroduplex; e) inserting the solid support into the slot of the electrophoretic gel under conditions sufficient to release all or a cleaved portion of the immobilized heteroduplex into the slot; and f) analyzing the released product by gel electrophoresis, the presence of a cleavage product being an indication of a mismatch in the test nucleic acid.

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