A kinetic model for subtractive hybridization.
Nucleic acid sequences that differ in abundance between two populations (target sequences) can be cloned by multiple rounds of subtractive hybridization and amplification by PCR. These sequences can be cDNAs representing up-regulated mRNAs, or genomic DNAs from deletion mutants. We have derived an equation that describes the recovery of such sequences, and have used this to simulate the outcome of up to 10 rounds of subtractive hybridization and PCR amplification. When the model was tested by comparing its predictions with the published results from genomic and cDNA subtractions, the predictions of the model were generally in good agreement with the published data. We have modelled the outcomes of genomic subtractions, for a variety of genomes, and have used it to compare various strategies for enriching targets. The model predicts that for genomes of less than 5 x 10(8) bp, deletions of as small as 1 kbp should represent > 99% of the DNA after three to six rounds of hybridization (depending on the enrichment procedure). As genomes increase in size, the kinetics of hybridization become an important limiting factor. However, even for genomes as large as 3 x 10(9) bp, it should be possible to isolate deletions of 5 kbp using the appropriate conditions. These simulations suggest that such methods offer a realistic alternative to chromosome walking for identifying genomic deletions for which there are known phenotypes, thereby considerably reducing time and effort. For cDNA subtractive hybridization, the model predicts that after six rounds of hybridization, sequences that do not differ in abundance between the tester and driver populations (the background) will represent < 1% of the subtracted population, and even quite modestly upregulated cDNAs should be successfully enriched. Where several up-regulated cDNAs are present, the predicted final representation is dependent on both the initial abundance and the degree of up-regulation.
Milner
J J
JJ
Plant Molecular Science Group, Institute of Biomedical and Life Sciences, Glasgow University.
Cecchini
E
E
Dominy
P J
PJ
D000818:Animals; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D005821:Genetic Techniques; D016678:Genome; D015894:Genome, Human; D006801:Humans; D007700:Kinetics; D008957:Models, Genetic; D009693:Nucleic Acid Hybridization; D010944:Plants / Q000235:genetics; D016133:Polymerase Chain Reaction; D017384:Sequence Deletion; D015854:Up-Regulation
D018076:DNA, Complementary; D018744:DNA, Plant