Automatable screening of yeast artificial-chromosome libraries based on the oligonucleotide-ligation assay.
The systematic screening of yeast artificial-chromosome (YAC) libraries is the limiting step in many physical mapping projects. To improve the screening throughput for a human YAC library, we designed an automatable strategy to identify YAC clones containing a specific segment of DNA. Our approach combines amplification of the target sequence from pooled YAC DNA by the polymerase chain reaction (PCR) with detection of the sequence by an ELISA-based oligonucleotide-ligation assay (OLA). The PCR-OLA approach eliminates the use of radioactive isotopes and gel electrophoresis, two of the major obstacles to automated YAC screening. Furthermore, the use of the OLA to test for the presence of sequences internal to PCR primers provides an additional level of sensitivity and specificity in comparison to methods that rely solely on the PCR.
Kwok
P Y
PY
Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110.
Gremaud
M F
MF
Nickerson
D A
DA
Hood
L
L
Olson
M V
MV
D001483:Base Sequence; D015139:Blotting, Southern; D015825:Chromosomes, Fungal; D004797:Enzyme-Linked Immunosorbent Assay; D015723:Gene Library; D015894:Genome, Human; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism*; D016133:Polymerase Chain Reaction
D009841:Oligonucleotides