A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones.
The recent development of yeast artificial chromosome (YAC) vectors has provided a system for cloning fragments that are over ten times larger than those that can be cloned in more established systems. We have developed a method for the rapid isolation of terminal sequences from YAC clones. The YAC clone is digested with a range of restriction enzymes, a common linker is ligated to the DNA fragments and terminal sequences are amplified using a vector specific primer and a linker specific primer. Sequence data derived from these terminal specific products can be used to design primers for a further round of screening to isolate overlapping clones. The method also provides a convenient method of generating Sequence Tagged Sites for the mapping of complex genomes.
Riley
J
J
ICI Pharmaceuticals, Biotechnology Department, Macclesfield, Cheshire, UK.
Butler
R
R
Ogilvie
D
D
Finniear
R
R
Jenner
D
D
Powell
S
S
Anand
R
R
Smith
J C
JC
Markham
A F
AF
D001483:Base Sequence; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D005784:Gene Amplification; D015723:Gene Library; D005821:Genetic Techniques; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics
D004262:DNA Restriction Enzymes