PROCEDURE FOR SUBTRACTIVE HYBRIDIZATION AND DIFFERENCE ANALYSIS

摘要

Improved methods for obtaining polynucleotides comprising sequences which differ between two populations of DNA or cDNA are provided. Improvements include reduction in the number of amplification cycles, use of a nuclease digestion step prior to amplification, a novel oligonucleotide adapter for the practice of the improved method, and novel methods for selective amplification of the desired unique fragments and selective degradation of fragments containing sequences common to both populations. Fragments of a sample population are amplified using a primer that endows the amplification products with resistance to nuclease degradation. Fragments of a control population are amplified using a primer that targets the amplification products for preferential degradation. Multiple cycles of hybridization, nuclease treatment and amplification are utilized to provide enrichment of fragments unique to the sample population. Such unique fragments may represent new or amplified sequences in the sample population, sequences that are differently arranged in the sample population compared to the control population, and sequences that are differentially expressed in a cDNA population. A variation of the technique also allows the isolation of fragments representing deletions in the sample population.