Use of a D17Z1 oligonucleotide probe for human DNA quantitation prior to PCR analysis of polymorphic DNA markers
- Roche Molecular Systems, Alameda, CA (United States); and others
The alpha-satellite DNA locus D17Z1 contains primate-specific sequences which are repeated several hundred times per chromosome 17. A probe that was designed to hybridize to a subset of the D17Z1 sequence can be used for very sensitive and specific quantitation of human DNA. Sample human genomic DNA is immobilized on nylon membrane using a slot blot apparatus, and then hybridized with a biotinylated D17Z1 oligonucleotide probe. The subsequent binding of streptavidin-horseradish peroxidase to the bound probe allows for either calorimetric (TMB) or chemiluminescent (ECL) detection. Signals obtained for sample DNAs are then compared to the signals obtained for a series of human DNA standards. For either detection method, forty samples can be quantitated in less than two hours, with a sensitivity of 150 pg. As little as 20 pg of DNA can be quantitated when using chemiluminescent detection with longer film exposures. PCR analysis of several VNTR and STR markers has indicated that optimal typing results are generally obtained within a relatively narrow range of input DNA quantities. Too much input DNA can lead to PCR artifacts such as preferential amplification of smaller alleles, non-specific amplification products, and exaggeration of the DNA synthesis slippage products that are seen with STR markers. Careful quantitation of human genomic DNA prior to PCR can avoid or minimize these problems and ultimately give cleaner, more unambiguous PCR results.
- OSTI ID:
- 134729
- Report Number(s):
- CONF-941009-; ISSN 0002-9297; TRN: 95:005313-1467
- 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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Related Subjects
BASIC STUDIES
44 INSTRUMENTATION
INCLUDING NUCLEAR AND PARTICLE DETECTORS
BIOLOGICAL MARKERS
DNA HYBRIDIZATION
OLIGONUCLEOTIDES
PROBES
POLYMERASE CHAIN REACTION
ACCURACY
HUMAN CHROMOSOME 17
DNA SEQUENCING
QUANTITATIVE CHEMICAL ANALYSIS
SENSITIVITY
PRIMATES
DNA
GENES
SAMPLE PREPARATION