Importance

Loading Importance Indicators...

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.

Loading the Abstract Image...

First Claim