| Autor: |
Matteson, Karla J, Urdea, Mickey S, Chung, Bon-Chu, Lim, Mu Lan, Miller, Walter L |
| Zdroj: |
Pediatric Research; April 1985, Vol. 19 Issue: 4 p250A-250A, 1p |
| Abstrakt: |
Short, chemically synthesized mixed-sequence oligonucleotides have been used to identify cDNA and gene clones. Recently, pairs of 19 to 23 base unique-sequence oligos differing at a single base have been used to identify disease-causing point mutations in human DNAs. Both techniques rely on the requirement of short oligonucleotides to have a perfect match with the probed gene in order to hybridize. We have used long oligonucleotides containing non-human sequences with unknown mismatches for the same purpose. Using a unique new DNA synthesizer (DNA 3:401, 1984) we produced a 63-base and 3 72-base oligonucleotides corresponding to various regions of the bovine cDNA for P450scc (20, 22 desmolase) the enzyme converting cholesterol to pregnenolone. All four oligos hybridize to Northern blots of human adrenal mRNA in the same pattern. Two of these oligonucleotides hybridize to Southern blots of normal human genomic DNA in identical patterns indicating the human genome contains a single P450scc gene. As the two non-hybridizing oligos hybridize to mRNA but not genomic DNA, they probably span introns. Southern blots of genomic DNA from 3 patients with 20, 22 desmolase deficiency (congenital lipoid adrenal hyperplasia) hybridize with the oligos in the same pattern as DNA from normals, implying the disease is not due to a detectable gene deletion. Advances in DNA synthesis may permit gene studies in human diseases without requiring the tedious intermediate cloning steps. |
| Databáze: |
Supplemental Index |
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