Further Characterization of the VNTR Probe LH1 (D5S110) and Applications for DNA Typing

Abstract

The single-locus probe LH1 (D5S110) originally described by Armour, J.P., et al, Genomics, Vol. 8, p.501, 1990 was evaluated for its effectiveness in DNA typing assays for human identification. A cloned, purified, 6-Kb DNA fragment containing sequences complementary to the D5S110 locus was labeled with ³²P-dCTP by the random primer method. LH1 appears to be one of the most sensitive single-locus probes observed, capable of detecting a DNA profile in 10 ng of Hae III restricted genomic DNA in a 24-hour autoradiographic exposure. An enzyme-linked oligonucleotide probe was constructed for use in a chemiluminescent hybridization and detection format. Nine repeats within the D5S110 cloned insert were sequenced, and a 30-base oligonucleotide was synthesized and labeled with alkaline phosphatase. The sensitivity and specificity of the alkaline phosphatase labeled oligonucleotide under the ACES 2.0 hybridization and detection conditions is comparable to that of the ³²P labeled LH1 probe. The enzyme-linked oligonucleotide probe was capable of detecting the correct banding pattern in 25 ng of Hae III restricted genomic DNA in a 3-hour exposure following a 3-hour ramp.