A random STS strategy for construction of YAC contigs spanning defined chromosomal regions.
Sequence tagged sites (STSs) that were generated via Alu-element-mediated polymerase chain reaction (Alu-PCR) and mapped to human Xq26 were used to isolate and overlap yeast artificial chromosomes (YACs). By collating the results of primary pool screening, the order of STSs and YACs was postulated directly. Subsequent isolation of 11 key YACs from 75 positive pools confirmed the proposed contig. Although only a small subset of the available Alu-PCR fragments was used, the STSs were generated at sufficient density to isolate all the YACs required and to identify all except one overlap directly. The results confirmed physical linkage of HPRT to DXS86 and DXS144E. Long-range continuity was determined purely by analysis of the 11 YAC colonies and required no end-rescue. This strategy is therefore an effective approach for the construction of YAC contigs spanning discrete chromosomal regions contained within somatic cell hybrids, with minimal prior knowledge of the region.
Cole
C G
CG
Paediatric Research Unit, United Medical School, Guy's Hospital, London, United Kingdom.
Dunham
I
I
Coffey
A J
AJ
Ross
M T
MT
Meier-Ewert
S
S
Bobrow
M
M
Bentley
D R
DR
D001483:Base Sequence; D002460:Cell Line; D002874:Chromosome Mapping; D015825:Chromosomes, Fungal; D015723:Gene Library; D015894:Genome, Human; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction; D016324:Sequence Tagged Sites; D014960:X Chromosome
D009841:Oligonucleotides