Subtractive hybridization with covalently binding homology

Details Subtractive hybridization with covalently binding homology Subtractive hybridization with covalently binding homology

1997-09-11

1999-07-27

Horlick, Kenneth R.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 912, C12Q 168, C12P 1934

Patent

active

059288728

ABSTRACT:
Excess amount of modified subtracter DNA from control cells is generated by carboxylating the base structures of its certain nucleotides with chemical agents in order to introduce covalent affinity between the modified subtracter and a non-modified tester DNA. Hybridization of the control subtracter and the experimental tester DNA is performed with a heat-melting and then cool-reassociation technique. While the desired different (heterologous) sequences remain in the form of hydrogen-binding, common (homologous) sequences of the hybridized DNA are covalently bonded to each other. Since the covalent bonding of the common sequences can not be broken during a polymerase chain reaction, resulting in no amplification of the common sequences but great amplification of the desired different sequences. The desired DNA sequences present after such covalent homologue subtraction and selective amplification represent those DNA sequences which only exist in the tester but not in the subtracter DNA library.

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