▪ Abstract Several unique properties of human mitochondrial DNA (mtDNA), including its high copy number, maternal inheritance, lack of recombination, and high mutation rate, have made it the molecule of choice for studies of human population history and evolution. Here we review the current state of knowledge concerning these properties, how mtDNA variation is studied, what we have learned, and what the future likely holds. We conclude that increasingly, mtDNA studies are (and should be) supplemented with analyses of the Y-chromosome and other nuclear DNA variation. Some serious issues need to be addressed concerning nuclear inserts, database quality, and the possible influence of selection on mtDNA variation. Nonetheless, mtDNA studies will continue to play an important role in such areas as examining socio-cultural influences on human genetic variation, ancient DNA, certain forensic DNA applications, and in tracing personal genetic history.

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Mitochondrial DNA and human evolution

Fluorescence-based sequencing is playing an increasingly important role in efforts to identify DNA polymorphisms and mutations of biological and medical interest. The application of this technology in generating the reference sequence of simple and complex genomes is also driving the development of new computer programs to automate base calling (Phred), sequence assembly (Phrap) and sequence assembly editing (Consed) in high throughput settings. In this report we describe a new computer program known as PolyPhred that automatically detects the presence of heterozygous single nucleotide substitutions by fluorescencebased sequencing of PCR products. Its operations are integrated with the use of the Phred, Phrap and Consed programs and together these tools generate a high throughput system for detecting DNA polymorphisms and mutations by large scale fluorescence-based resequencing. Analysis of sequences containing known DNA variants demonstrates that the accuracy of PolyPhred with single pass data is >99% when the sequences are generated with fluorescent dye-labeled primers and approximately 90% for those prepared with dye-labeled terminators.

/pdf/polyphred-automating-the-detection-and-genotyping-of-single-2qosurowau.pdf

PolyPhred: automating the detection and genotyping of single nucleotide substitutions using fluorescence-based resequencing

Reports of single base-pair mutations within gene coding regions causing human genetic disease were collated. Thirty-five per cent of mutations were found to have occurred within CpG dinucleotides. Over 90% of these mutations were C → T or G → A transitions, which thus occur within coding regions at a frequency 42-fold higher than that predicted from random mutation. These findings are consistent with methylation-induced deamination of 5-methyl cytosine and suggest that methylation of DNA within coding regions may contribute significantly to the incidence of human genetic disease.

The CpG dinucleotide and human genetic disease

https://www.cell.com/cell/pdf/0092-8674(87)90158-9.pdf

A genetic linkage map of the human genome

Oligonucleotide microarray (DNA chip)–based hybridization analysis is a promising new technology which potentially allows rapid and cost–effective screens for all possible mutations and sequence variations in genomic DNA. Here, I review current strategies and uses for DNA chip–based resequencing and mutational analysis, the underlying principles of experimental designs, and future efforts to improve the sensitivity and specificity of chip–based assays.

Resequencing and mutational analysis using oligonucleotide microarrays

Fifteen different restriction fragment length polymorphisms (RFLPs) were detected in the human genome using 19 cloned DNA segments, derived from flow-sorted metaphase chromosomes or total genomic DNA, as hybridization probes. Since these clones were selected at random with respect to their coding potential, their analysis permitted an unbiassed estimate of single-copy DNA sequence heterozygosity in the human genome. Since our estimate (h=0.0037) is an order of magnitude higher than previous estimates derived from protein data, most of the polymorphic variation present in the genome must occur in non-coding sequences. In addition, it was confirmed that enzymes containing the dinucleotide CpG in their recognition sequence detect more polymorphic variation than those that do not contain CpG.

Barbara A. Smith

Papers

An estimate of unique DNA sequence heterozygosity in the human genome.