A novel and rapid method for isolating sequences adjacent to rare cutting sites and their use in physical mapping.
We describe a simple PCR based technique which can be used to isolate sequences adjacent to rare cutter sites and can subsequently be employed for the construction of long range physical maps. The method involves the ligation of an adaptor to rare cutter sequences and its use as a target for forward priming in PCR. Primers to Alu repeat elements initiate synthesis of the reverse strand. Using this technique any rare cutter site which has a repeat element within amplification range can be cloned. We have isolated six unique sequences around NotI sites from an irradiation reduced hybrid containing a fragment of human chromosome 22 and are using these for physical mapping around the Ewing's sarcoma translocation breakpoint on chromosome 22.
Patel
K
K
Human Cytogenetics Laboratory, ICRF, London, UK.
Cox
R
R
Shipley
J
J
Kiely
F
F
Frazer
K
K
Cox
D R
DR
Lehrach
H
H
Sheer
D
D
D001483:Base Sequence; D002478:Cells, Cultured; D002874:Chromosome Mapping / Q000379:methods*; D002892:Chromosomes, Human, Pair 22; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000235:genetics; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D006801:Humans; D006822:Hybrid Cells; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D012512:Sarcoma, Ewing / Q000235:genetics; D014178:Translocation, Genetic / Q000235:genetics; D014407:Tumor Cells, Cultured
D004277:DNA, Single-Stranded; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases