TY - JOUR
T1 - Kilo-sequencing
T2 - An ordered strategy for rapid DNA sequence data acquisition
AU - Barnes, Wayne M.
AU - Bevan, Michael
N1 - Funding Information:
We thank M.-D.Chilton for subcloned Ti-DNA, Paula Son and Elodee Tuley for technical assistance, G.Gallupi (Monsanto) and N.E.Biolabs for gifts of primer, and the NIH (grant no. GR24956) and the American Cancer Society (Faculty Research Award to W.B.) for support.
PY - 1983/1/25
Y1 - 1983/1/25
N2 - A strategy for rapid DNA sequence acquisition in an ordered, nonrandom manner, while retaining all of the conveniences of the dideoxy method with M13 transducing phage DNA template, is described. Target DNA 3 to 14 kb in size can be stably carried by our M13 vectors. Suitable targets are stretches of DNA which lack an enzyme recognition site which is unique on our cloning vectors and adjacent to the sequencing primer; current sites that are so useful when lacking are Pst, Xba, HindIII, BglII, EcoRI. By an in vitroprocedure, we cut RF DNA once randomly and once specifically, to create thousands of deletions which start at the unique restriction site adjacent to the dideoxy sequencing primer and extend various distances across the target DNA. Phage carrying a desired size of deletions, whose DNA as template will give rise to DNA sequence data in a desired location along the target DNA , may be purified by electophoresis alive on agarose gels . Phage running in the same location on the agarose gel thus conveniently give rise to nucleotide sequence data from the same kilobase of target DNA.
AB - A strategy for rapid DNA sequence acquisition in an ordered, nonrandom manner, while retaining all of the conveniences of the dideoxy method with M13 transducing phage DNA template, is described. Target DNA 3 to 14 kb in size can be stably carried by our M13 vectors. Suitable targets are stretches of DNA which lack an enzyme recognition site which is unique on our cloning vectors and adjacent to the sequencing primer; current sites that are so useful when lacking are Pst, Xba, HindIII, BglII, EcoRI. By an in vitroprocedure, we cut RF DNA once randomly and once specifically, to create thousands of deletions which start at the unique restriction site adjacent to the dideoxy sequencing primer and extend various distances across the target DNA. Phage carrying a desired size of deletions, whose DNA as template will give rise to DNA sequence data in a desired location along the target DNA , may be purified by electophoresis alive on agarose gels . Phage running in the same location on the agarose gel thus conveniently give rise to nucleotide sequence data from the same kilobase of target DNA.
UR - http://www.scopus.com/inward/record.url?scp=0021111540&partnerID=8YFLogxK
U2 - 10.1093/nar/11.2.349
DO - 10.1093/nar/11.2.349
M3 - Article
C2 - 6298723
AN - SCOPUS:0021111540
VL - 11
SP - 349
EP - 368
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 2
ER -