Vectorettes for long chromosome walking in genomic DNA of the human p53 gene

Abstract

Chromosome walking in mammalian DNA by vectorette PCR is not always very specific, and the walks have been limited to distances <1 kb. To improve the method, we have designed new vectorettes, which unlike the currently used ones have very little repetitive sequences or homology with known DNA sequences of various origins in the data banks. We have tested these new vectorettes for chromosome walking in human p53 tumor suppressor gene, human tissue-type plasminogen activator gene, and mouse stanniocalcin gene with good success. In chromosome walking of the human p53 gene, we isolated gene-specific fragments of 2.4. kb, and by walking in a bacterial artificial chromosome (BAC) clone carrying the mouse stanniocalcin gene, we isolated fragments up to about 7 kb in size. We further sequenced the 5′ region of the p53 gene and found that the nucleotides upstream of −1009 are transcribed in antisense orientation into a messenger RNA (mRNA) (flj10385) encoding a putative serine/threonine kinase.