DNA fingerprinting and forensic medicine.

In forensic medicine, DNA fingerprinting for identification is becoming a necessary procedure. A method to radiolabel M13 DNA probe by primer extension using a specific oligonucleotide primer was developed. The method specifically labeled the two 15 bp repeats in M13 DNA which hybridize to target DNA giving rise to DNA fingerprinting patterns. The M13 probe labeled by this method has proven useful for individual identification, paternity testing and monitoring reconstitution in bone marrow transplantation. The genetic locus D1S80 and D17S30 containing a variable number of tandem repeats (VNTR) have also been successfully amplified from human genomic DNA isolated from blood (50 ng from each sample) by the polymerase chain reaction (PCR) using oligonucleotide primers complementary to the flanking sequences as primers for amplification. DNA bands were detected by ethidium bromide staining after electrophoresis on agarose gels. Analysis of this VNTR locus was thus achieved without the need for Southern blot or radioactive material. The small size of the DNA fragments produced in the PCR amplification permited good resolution of individual alleles. The precise specification of the number of tandem repeats present in each allelic fragment was reproducible from one analysis to another.

Field evaluation of simplified radioactive and nonradioactive DNA probe methods for the diagnosis of falciparum malaria.

Specific DNA probe hybridization technique is one method of choice for detection of malaria infection. It provides an obvious advantage over conventional microscopy when large numbers of samples are simultaneously monitored. The method was simplified so that preparation and processing of blood specimens were all performed on membrane filters. Background signals generated from blood components were removed by treating samples spotted on the membrane with a series of buffer washes without the necessity of a protease digestion step. Hybridization was monitored using either 32P-or digoxigenin-labeled DNA probe. 849 field samples collected from various malaria endemic areas in Thailand have been evaluated by this protocol and compared with microscopic examination. Sensitivity obtained by this procedure was comparable to that of microscopy at a malaria clinic. The specificities of both types of DNA probes were better than 93%, but digoxigenin-labeled probe performed better than 32P-labeled one when the numbers of parasites were less than 25 per 200 white blood cells.

Detection of Plasmodium falciparum using a cloned DNA probe: a simple procedure suitable for field application.

A simple procedure was developed for spotting blood samples directly onto nylon membrane filter, without the necessity to treat samples with pronase or proteinase K, followed by hybridizing with 32P-labelled DNA probe, pUNK1-45. This probe detected specifically P. falciparum DNA and did not cross react with DNA from man, P. knowlesi, P. chabaudi or P. cynomolgi. The probe was sensitive to detect a parasitemia of 0.001% in 20 microliters of blood.