A method for difference cloning: gene amplification following subtractive hybridization

Wieland, I., Bolger, G., Asouline, G., Wigler, M. H. (April 1990) A method for difference cloning: gene amplification following subtractive hybridization. Proc Natl Acad Sci U S A, 87 (7). pp. 2720-4. ISSN 0027-8424

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URL: https://www.ncbi.nlm.nih.gov/pubmed/2138783
DOI: 10.1073/pnas.87.7.2720


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.

Item Type: Paper
Uncontrolled Keywords: Bacteriophage lambda genetics Base Sequence Cell Line Cloning Molecular methods DNA genetics isolation & purification DNA, Viral genetics Female Humans Molecular Sequence Data Nucleic Acid Amplification Techniques Nucleic Acid Hybridization Oligonucleotide Probes Placenta Polymerase Chain Reactionmethods Pregnancy
Subjects: Investigative techniques and equipment > cloning
Investigative techniques and equipment > assays > cloning
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > genes, structure and function > gene amplification
CSHL Authors:
Communities: CSHL labs > Wigler lab
Depositing User: CSHL Librarian
Date: April 1990
Date Deposited: 06 Apr 2012 14:45
Last Modified: 09 Nov 2017 20:44
Related URLs:
URI: https://repository.cshl.edu/id/eprint/25979

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