Direct analysis of single nucleotide variation in human DNA and RNA using in situ dot hybridization.
Using oligonucleotide hybridization, single and multiple nucleotide differences between alleles were detected directly in genomic DNA without electrophoretic separation. The DNA was immobilized in depressions in an agarose gel (in situ dots) and hybridized with radiolabeled, allele-specific oligonucleotide probes. An oligonucleotide complementary to a unique sequence region of the human major histocompatibility complex gene HLA-B27 only hybridized with genomic DNA from an HLA-B27-positive individual. Two other oligonucleotides complementary to the normal human beta-globin gene (beta A) and to the sickle cell globin gene (beta S) were synthesized. Using competition hybridization conditions which included the presence of a 10-fold molar excess of unlabeled oligonucleotide complementary to the other beta-globin allele, DNA from individuals homozygous for the normal beta-globin gene (beta A beta A) hybridized to the beta A probe exclusively, whereas DNA from individuals homozygous for the sickle cell globin gene (beta S beta S) hybridized only with the probe for the sickle cell gene. As expected, DNA from heterozygous individuals bound to both probes. Similar results were obtained with total human RNA immobilized in in situ dots. Possible applications of this methodology include genetic disease diagnosis, population carrier screening, HLA "DNA" typing, and DNA and RNA sequence polymorphism analysis.
Wu
D Y
DY
Department of Molecular Biochemistry, Beckman Research Institute of the City of Hope, Duarte, CA 91010.
Nozari
G
G
Schöld
M
M
Conner
B J
BJ
Wallace
R B
RB
D004247:DNA / Q000378:metabolism*; D006801:Humans; D009693:Nucleic Acid Hybridization; D012313:RNA / Q000378:metabolism*
D012313:RNA; D004247:DNA