Cloning the ends of size selected Sfi I fragments.
As an initial step in the physical mapping of the fragile X region a library of Sfi I ends was constructed from the size class of human Sfi I DNA fragments, which includes the fragment with the locus DXS105. Since Sfi I recognizes the sequence GGCCNNNNNGGCC and leaves a 3 base indeterminate "sticky" end, we used a mixture of 64 synthetic deoxynucleotide oligomers to modify these ends for cloning. The oligomers were of the general form AATTNNN. Ligation of these heptamers to the indeterminate Sfi I ends converted them to the EcoR I sticky end. A suppressor tRNA gene was ligated onto this end as a selectable marker and the DNA was cloned in the lambda phage vector Charon 21A. Analysis showed that clones selected for the presence of the tRNA gene contained Sfi I ends. Because this library was constructed from a specific size class of fragments, it was very reduced in complexity. This will simplify the process of identifying the clone which contains the end of the DXS105 fragment. The use of this strategy for chromosome "jumping" is discussed.
Nolin
S L
SL
Department of Human Genetics, NYS Institute for Basic Research in Developmental Disabilities, Staten Island 10314.
Dobkin
C S
CS
D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific; D016153:Genes, Suppressor; D005813:Genes, Synthetic; D005819:Genetic Markers; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D012641:Selection, Genetic
D005819:Genetic Markers; D009838:Oligodeoxyribonucleotides; D004247:DNA; C041749:endodeoxyribonuclease SfiI; D015252:Deoxyribonucleases, Type II Site-Specific