Nucleotide analog-containing hybrid subtraction with sequentiall

Details Nucleotide analog-containing hybrid subtraction with sequentiall Nucleotide analog-containing hybrid subtraction with sequentiall

1997-05-12

1999-02-16

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

058719270

ABSTRACT:
The present invention provides a method for fast, simple, and reliable isolation of desired different sequences from two DNA libraries. Excess amount of nucleotide analog-containing DNA subtracter from control cells is generated by incorporating nucleotide analog with a template-dependent extension reaction to introduce susceptible-sites for subsequent enzymatic digestion. Hybridization of the control subtracter and experimental DNA is performed with a heat-melting and then cool-reassociation technique. The hybridized DNAs are subtracted with nucleotide analog-removing enzyme first, resulting in nicking or gapping all nucleotide analog-containing hybrid duplexes which are further digested by single-strand-specific nuclease. Desired DNA sequences from the experimental cells, but not the control ones stay intact throughout the digestion procedure and can be selectively amplified at the end. This technique is designed for the subtractive hybridization of different sequences between two DNA libraries from distinct cell sources and will allow more efficient isolations in experiments on cancer formation, development of gene therapy, and understanding of pathological status and developmental regulation.

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