Dinucleotide repeat polymorphisms isolated by the polymerase chain reaction.

DNA sequences containing tandem dinucleotide repeats represent an abundant source of DNA polymorphism in human and other eukaryotic genomes. Here we describe a novel technique for the identification and characterization of regions of DNA containing these repetitive elements. Using primers designed to recognize tandem dinucleotide repeat sequences and limiting dilution of a target genomic library enable amplification by polymerase chain reaction (PCR) of single-target molecules containing dinucleotide repeats. Amplified material was sequenced by the PCR direct method and by the resultant sequences used to design locus-specific primers. This study identified and characterized four anonymous dinucleotide repeat sequences, three of which exhibited polymorphism. Although developed for dinucleotide repeats, the technique is universally applicable to repeat DNA elements of a size usually analyzed by PCR. The technique is comparatively rapid, eliminates library screening and its associated manipulations, and compares favorably with existing methods for the recovery of repetitive DNAs.

In vivo human somatic mutation: frequency and spectrum with age.

The number and molecular nature of in vivo mutations in relation to age was studied at the autosomal HLA-A locus in human lymphocytes. Mutant lymphocytes were isolated by immunoselection, cloned at limiting dilution and enumerated, and the HLA-A gene and other polymorphic gene loci on chromosome 6 were studied by Southern blotting to determine gene dosage and loss of heterozygosity. Results of 167 assays in 73 individuals showed that the total number of mutant lymphocytes increased significantly with age from a geometric mean frequency of 0.71 x 10(-5) in neonates to 6.53 x 10(-5) in elderly individuals. Analysis of rearrangement of T lymphocyte receptor beta or gamma chain genes gave a best estimate of 3.3% for the proportion of mutant lymphocytes detected which are clonally related. Molecular study of 434 mutants from 31 individuals showed no change on Southern blotting in 64.7%, gene deletion in 2.8% and mitotic recombination in 32.5%. Two mutants due to gene conversion but no mutants due to non-disjunction were detected. The number of 'no change' and recombination mutants increased significantly with age. There was a significant difference between individuals in the proportion of mutants which resulted from mitotic recombination and the data suggested that the proportion was bimodally distributed. The point of crossing-over in recombination mutants was predominantly randomly distributed between the HLA-A locus and the centromere.

Molecular nature of in vivo mutations in human cells at the autosomal HLA-A locus.

The mutations present in vivo in normal human cells were studied at the HLA-A locus by isolating mutant lymphocytes using antibody-complement immunoselection and cloning at limiting dilution. The molecular basis for mutation in 127 mutant lymphocytes from 10 individuals was determined by studying a variety of polymorphic gene loci on both arms of chromosome 6. No change was detected in 78 mutants (61.4%), gene deletion was detected in 11 (8.7%), and mitotic recombination was detected in 38 (29.9%). Neither gene conversion nor chromosome loss was detected. These observations document the mechanisms responsible for gene loss in normal human cells in vivo, emphasize the importance of mitotic recombination, and indicate the similarity between mutational mechanisms in normal cells and in cancer cells.

Mutations in human lymphocytes commonly involve gene duplication and resemble those seen in cancer cels.

Mutations in human lymphocytes are commonly due to gene deletion. To investigate the mechanism of deletion for autosomal genes, we immunoselected lymphocytes mutated at the HLA-A locus and cloned them for molecular analysis. Of 36 mutant clones that showed deletion of the selected HLA-A allele, 8 had resulted from a simple gene deletion, whereas 28 had resulted from a more complex mutational event involving reduplication of the nonselected HLA-A allele as indicated by hybridization intensity on Southern blots. In 3 of the 28 clones, retention of heterozygosity at the HLA-B locus indicated that the reduplication was due to recombination between the two chromosomes 6; but in the remaining 25 clones, distinction could not be made between recombination and chromosome reduplication. The results indicate that mutations in normal somatic cells frequently result in hemizygosity or homozygosity at gene loci and, thereby, resemble the mutations thought to be important in the etiology of various forms of cancer.