A method for difference cloning: gene amplification following subtractive hybridization.
basic_science · Level V
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- Record sourced from PubMed, PMID 2138783.
- Also identified by PMC identifier 53762.
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Abstract
We describe a procedure for genomic difference cloning, a method for isolating sequences present in one genomic DNA population ("tester") that is absent in another ("driver"). By subtractive hybridization, a large excess of driver is used to remove sequences common to a biotinylated tester, enriching the "target" sequences that are unique to the tester. After repeated subtractive hybridization cycles, tester is separated from driver by avidin/biotin affinity chromatography, and single-stranded target is amplified by the polymerase chain reaction, rendering it double-stranded and clonable. We model two situations: the gain of sequences that result from infection with a pathogen and the loss of sequences that result from a large hemizygous deletion. We obtain 100- to 700-fold enrichment of target sequences.
Medical subject headings
- Cloning, Molecular
- DNA
- Nucleic Acid Amplification Techniques
- Nucleic Acid Hybridization
- Polymerase Chain Reaction