A method for difference cloning: gene amplification following subtractive hybridization.
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.
Wieland
I
I
Cold Spring Harbor Laboratory, NY 11724.
Bolger
G
G
Asouline
G
G
Wigler
M
M
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004279:DNA, Viral / Q000235:genetics; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010920:Placenta; D016133:Polymerase Chain Reaction / Q000379:methods*; D011247:Pregnancy
D004279:DNA, Viral; D015345:Oligonucleotide Probes; D004247:DNA