Highly efficient method for obtaining a subtracted genomic DNA library by the modified in-gel competitive reassociation method.
A highly efficient method to obtain a subtracted genomic DNA library using 1 microgram of target DNA was developed by modification of the previously reported in-gel competitive reassociation procedure. The modified method was based on polymerase chain reaction amplification after selective purification of a target-target reassociated molecule of subtracted DNA fragments to increase cloning efficiency. For a model experimental system, the subtracted DNA library was constructed after two cycles of subtractive reassociation between cervical cancer DNA fragments containing human papilloma virus DNA and the 100-fold excess of dephosphorylated normal tissue DNA fragments which were size-fractionated in agarose gel. Colony hybridization using human papilloma virus DNA as a probe revealed that a more than 500-fold enrichment of human papilloma virus DNA sequences in the subtracted DNA library could easily be obtained. This simple and efficient method will enable us to isolate an unknown foreign DNA fragment and an unknown amplified DNA fragment which might be present in cancer.
Genetics Division, National Cancer Center Research Institute, Tokyo, Japan.
H
H
Sasaki
S
S
Nomura
N
N
Akiyama
A
A
Takahashi
T
T
Sugimura
M
M
Oishi
M
M
Terada
D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004273:DNA, Neoplasm / Q000302:isolation & purification*; D004279:DNA, Viral / Q000302:isolation & purification*; D005260:Female; D015698:Genomic Library; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D027383:Papillomaviridae / Q000235:genetics; D010920:Placenta / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000821:virology
D004273:DNA, Neoplasm; D004279:DNA, Viral