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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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.
| 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 |
| PMCID: | PMC53762 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/25979 |
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