DNA profiling of extended tracts of primitive DNA repeats: Direct identification of unstable simple repeat loci in complex genome
- Univ. of Toronto (Canada); and others
The most simple DNA repetitive elements, with repetitive monomer units of only 1-10 bp in tandem tracts, are an abundant component of the human genome. The expansion of at least one type of these repeats ((CCG)n and (CTG)n) have been detected for a several neurological diseases with anticipation in successive generations. We propose here a simple method for the identification of particularly expanded repeats and for the recovery of flanking sequences. We generated DNA probes using PCR to create long concatamers (n>100) by amplification of the di-, tri-, tetra-, penta- and hexa-nucleotide repeat oligonucleotide primer pairs. To reduce the complexity of the background band pattern, the genomic DNA was restricted with a mixture of at least five different endonucleases, thereby reducing the size of restriction fragments containing short simple repeat arrays while leaving intact the large fragments containing the longer simple repeats arrays. Direct blot hybridization has shown different {open_quotes}DNA fingerprint{close_quotes} patterns with all arbitrary selected di-hexa nucleotide repeat probes. Direct hybridization of the (CTG)n and (CCG)n probes revealed simple or multiple band patterns depending upon stringency conditions. We were able to detect the presence of expanded unstable tri-nucleotide alleles by (CCG)n probe for some FRAXA subjects and by (CTG)n probe for some myotonic dystrophy subjects which were not present in the parental DNA patterns. The cloning of the unstable alleles for simple repeats can be performed by direct recover from agarose gels of the aberrant unstable bands detected above. The recovered flanking regions can be cloned, sequenced and used for PCR detection of expanded alleles or can be used to screen cDNA. This method may be used for testing of small families with diseases thought to display clinical evidence of anticipation.
- OSTI ID:
- 133866
- Report Number(s):
- CONF-941009-; ISSN 0002-9297; TRN: 95:005313-0600
- Journal Information:
- American Journal of Human Genetics, Vol. 55, Issue Suppl.3; Conference: 44. annual meeting of the American Society of Human Genetics, Montreal (Canada), 18-22 Oct 1994; Other Information: PBD: Sep 1994
- Country of Publication:
- United States
- Language:
- English
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