Assessment of PCR of the D17S30 locus for forensic identification.

PCR analysis of the VNTR locus D17S30 was assessed for its potential use in forensic identification analysis. "Allelic drop-out," the inefficient amplification of some alleles, complicates the interpretation of DNA typing at this locus. PCR conditions were varied in an effort to improve amplification of the alleles at this locus. Such changes included the use of denaturants, formamide and DMSO, to overcome any incomplete denaturation of template strands due to GC content or allele size. Lowering the annealing temperature during the PCR cycle enhanced the amplification of a larger fragment, but this was not related to the D17S30 locus. It appears that the structure of the genome of some individuals rendered PCR amplification inefficient at this locus.

Direct sequencing of double-stranded PCR products incorporating a chemiluminescent detection procedure.

A simple and reliable method is described for direct sequencing of material generated by the polymerase chain reaction (PCR). Sequencing reactions can be performed directly on PCR products without the need for purification of the template by removal of residual deoxyribonucleoside triphosphates or primers. The coupling of a chemiluminescent detection system with the use of the same primers in the initial and sequencing PCR's allows for sequencing of a number of PCR products on the one gel.

Purification of human leucocyte DNA: proteinase K is not necessary.

A rapid nontoxic method for the purification of DNA from human leucocytes is described. Preliminary experiments which tested different methods of DNA purification indicated that digestion of proteins with proteinase K was unnecessary. This led to the development of a simple procedure involving lysis of the cells in SDS followed by extraction with 6 M NaCl. The method described overcomes the requirement for lengthy incubations in the presence of expensive proteinase K and subsequent extraction with toxic chemicals.

Circular mitochondrial DNA molecules from petite mutants of Saccharomyces cerevisiae: resolution by polyacrylamide gel electrophoresis.

Mitochondrial DNA (mtDNA) from petite strain K45 of Saccharomyces cerevisiae contains about 7% circular DNA molecules which comprise a simple oligomeric series based on a monomeric size of 1.7 kilobase pairs. Electrophoresis of K45 mtDNA on a polyacrylamide-agarose slab gel fractionates the mtDNA into a major band (containing linear DNA) and several faster running minor bands each containing particular size class of circular DNA molecules. From study of mtDNA from K45 and two other simple petites it was found that the mobility of circles is inversely proportional to the logarithm of the circle size. Polyacrylamide gel electrophoresis thus permits the separation of circular mtDNA from the linear mtDNA of simple petites, and physically resolves circles of different size from one another.

Role of mitochondria in the origin of chloroplast starch grains: description of the phenomenon.

By phase microscopic observation of living palisade parenchyma cells in sections of Nicotiana excelsior leaves from plants previously placed in the dark for 72 hours, 30 to 45 minutes of light is found to induce mitochondria to remain stationary within the concavity of the chloroplasts and become round. Extending the illumination period to 60 to 90 minutes causes the stationary mitochondria in the concavity to change from a translucent to an opaque appearance, the change coinciding with the first appearance of starch as detected by blue staining of the grains with I(2)-KI. It is speculated that an interaction bearing some resemblance to the previously described interaction between mitochondria and the mobile phase of the chloroplasts may also operate in the starch grain phenomenon.