An improved pulsed-field polyacrylamide gel electrophoresis system for physical selection of linking clones: isolation of SfiI linking clones from a chromosome 21-specific library.
We had previously developed an efficient procedure for selective cloning of rare-cutter linking fragments that is based on physical separation of linking clone DNAs by pulsed-field polyacrylamide gel electrophoresis (PF-PAGE). An advantage of the physical selection procedure over the conventional cloning-based ones utilizing the insertion of selection marker or vector sequences into the rare-cutter sites is that it can be readily applied to the selection of linking fragments for rare-cutters, generating ambiguous cohesive end sequences such as SfiI (GGCCNNNN/NGGCC). In the present work, the physical separation procedure was improved by introducing a discontinuous buffer system into PF-PAGE, and its feasibility was exemplified by the selective isolation of SfiI linking clones from a human chromosome 21-specific library. This simple and efficient procedure will provide a useful tool for genome analysis.
Ito
T
T
Department of Preventive Medicine, Nagasaki University, Japan.
Ohgusu
H
H
Oishi
N
N
Sakaki
Y
Y
D001483:Base Sequence; D002891:Chromosomes, Human, Pair 21; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism; D004270:DNA, Circular / Q000302:isolation & purification; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D015723:Gene Library; D008040:Genetic Linkage; D005821:Genetic Techniques; D006801:Humans; D008969:Molecular Sequence Data
D004270:DNA, Circular; D004247:DNA; C041749:endodeoxyribonuclease SfiI; D015252:Deoxyribonucleases, Type II Site-Specific