MITOCHONDRIAL D-LOOP SIGNATURES PRODUCED BY LOW-STRINGENCY SINGLE SPECIFIC PRIMER PCR CONSTITUTE A SIMPLE COMPARATIVE HUMAN IDENTITY TEST

We have developed a technique called ''LSSP-PCR'' (low-stringency sing le specific primer PCR) that detects single or multiple mutations in D NA. A purified DNA fragment is submitted to PCR by using a single prim er specific for one of the extremities of the fragment, under conditio ns of very low stringency. The primer hybridizes specifically to its c omplementary extremity and nonspecifically to multiple sites within th e fragment, in a sequence-dependent manner. A complex set of reaction products is thus created that, when separated by electrophoresis, cons titutes a unique ''gene signature.'' We here report the application of LSSP-PCR to the detection of sequence variation in the control (D-loo p) region of human mtDNA, which is known to differ significantly betwe en unrelated individuals. We prepared human DNA samples from blood and amplified a 1,024-bp portion of the mtDNA control region, using prime rs 1,15996 and H408. The amplified mtDNA fragments were then reamplifi ed under LSSP-PCR conditions by using 1,15996 or H408 as drivers to pr oduce complex signatures that always differed between unrelated indivi duals and yet were highly reproducible. In contrast, all mother-child pairs tested were identical, as expected from the matrilineal inherita nce of mtDNA. Thus, the use of LSSP-PCR to produce D-loop signatures c onstitutes a powerful new technique for mtDNA-based comparative identi ty testing.