Systematic screening of yeast artificial-chromosome libraries by use of the polymerase chain reaction.
We have developed an approach for screening ordered arrays of yeast artificial-chromosome (YAC) clones containing human DNA that is based on the polymerase chain reaction (PCR). This approach is designed to determine the locations of positive clones within a YAC library that is stored as individual clones in 96-well microtiter plates. The high sensitivity and specificity of the PCR allow the detection of target sequences in DNA prepared from pools of 1920 or more YAC clones. The PCR-based screening protocol is performed in two successive stages, which effectively limit the location of a positive clone to four microtiter plates (384 clones). Final localization of each positive clone is accomplished by conventional DNA.DNA hybridization using a single filter containing the YAC clones from the appropriate four microtiter plates. This PCR-based screening strategy has proven highly efficient, allowing the identification and isolation of numerous YAC clones containing specific human genes. The prospects of developing a strategy for screening YAC libraries based completely on PCR assays are discussed, as are the potential applications of this approach to the systematic analysis of the human genome.
Department of Genetics, Washington University School of Medicine, Saint Louis, MO 63110.
E D
ED
Green
M V
MV
Olson
D001483:Base Sequence; D015825:Chromosomes, Fungal; D002877:Chromosomes, Human; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D015723:Gene Library; D005796:Genes; D015894:Genome, Human; D006801:Humans; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics
D015345:Oligonucleotide Probes; D004247:DNA