Whole genome PCR: application to the identification of sequences bound by gene regulatory proteins.
A strategy is described that allows the isolation of DNA sequences that can bind to gene regulatory proteins. Total genomic DNA is first converted to a form that is suitable for amplification by the polymerase chain reaction (Whole Genome PCR), and the DNA sequences of interest are selected by binding to the regulatory protein and immune precipitation. Because sequences recovered from the selection step can be amplified by PCR, the selection process can be designed for maximum enrichment with little concern about recovery. Furthermore, the selection process can be repeated as often as necessary. Sequences recovered after amplification can be cloned and/or used as hybridization probes. As a test of this strategy, we selected human sequences that bound to Xenopus transcription factor IIIA (TFIIIA). Seven clones were isolated that were on the average 94% identical to the previously described 61 bp binding site of TFIIIA. This strategy could be adapted to isolate sequences that can be selected by any physical or biological method.
Kinzler
K W
KW
Oncology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21231.
Vogelstein
B
B
D001483:Base Sequence; D015139:Blotting, Southern; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase; D005784:Gene Amplification; D005796:Genes; D005809:Genes, Regulator; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D012341:RNA, Ribosomal, 5S / Q000235:genetics; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors / Q000378:metabolism*
D012341:RNA, Ribosomal, 5S; D035582:Transcription Factor TFIIIA; D014157:Transcription Factors; D004247:DNA; D004259:DNA-Directed DNA Polymerase