Detection of DNA sequence polymorphisms in human genomic DNA by using denaturing gradient gel blots.
Denaturing gradient gel electrophoresis can detect sequence differences outside restriction-enzyme recognition sites. DNA sequence polymorphisms can be detected as restriction-fragment melting polymorphisms (RFMPs) in genomic DNA by using blots made from denaturing gradient gels. In contrast to the use of Southern blots to find sequence differences, denaturing gradient gel blots can detect differences almost anywhere, not just at 4-6-bp restriction-enzyme recognition sites. Human genomic DNA was digested with one of several randomly selected 4-bp recognition-site restriction enzymes, electrophoresed in denaturing gradient gels, and transferred to nylon membranes. The blots were hybridized with radioactive probes prepared from the factor VIII, type II collagen, insulin receptor, beta 2-adrenergic receptor, and 21-hydroxylase genes; in unrelated individuals, several RFMPs were found in fragments from every locus tested. No restriction map or sequence information was used to detect RFMPs. RFMPs can be used as genetic markers, because their alleles segregate in a Mendelian manner. Unlike most other methods for detecting DNA sequence polymorphisms, a genomic DNA blot made from one gel can be hybridized consecutively with many (30 or more) different probes.
Gray
M R
MR
Department of Obstetrics and Gynecology, Tufts University School of Medicine, Boston, MA 02111.
D001345:Autoradiography; D015139:Blotting, Southern; D003094:Collagen / Q000235:genetics; D004247:DNA / Q000235:genetics*; D015342:DNA Probes; D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000235:genetics; D006801:Humans; D009691:Nucleic Acid Denaturation; D010375:Pedigree; D011110:Polymorphism, Genetic; D011972:Receptor, Insulin / Q000235:genetics; D011943:Receptors, Adrenergic, beta / Q000235:genetics; D013255:Steroid 21-Hydroxylase / Q000235:genetics
D015342:DNA Probes; D011943:Receptors, Adrenergic, beta; D005169:Factor VIII; D003094:Collagen; D004247:DNA; D013255:Steroid 21-Hydroxylase; D011972:Receptor, Insulin