Correct heteroduplex formation for mutation detection analysis.

BACKGROUND: The majority of mutation detection methods for unknown mutations are polymerase chain reaction (PCR)-based methods dependent on the formation of heteroduplexes between wild-type and mutant strands of DNA. METHODS AND RESULTS: This report discusses the difficulties associated with forming heteroduplexes with a large DNA fragment and the implications for subsequent mutation detection by the chemical cleavage of mismatch technique and other methods reliant on heteroduplex formation. It was found that the size and sequence context of the fragment being investigated inhibited correct heteroduplex formation. The problem was overcome by dividing the sequence into two overlapping fragments. CONCLUSIONS: Early identification of this problem in other fragments will help with the rapid optimization of PCR-based mutation detection methods.

New MspI polymorphism at +83 bp of the human apolipoprotein AI gene: association with increased circulating high density lipoprotein cholesterol levels.

We recently showed that loss of a MspI restriction site in the 5'-end (intron 1) of the apolipoprotein (apo) AI gene is due to a C to T transition (+83 bp) and/or a G to A transition (+84 bp). Since this region may be relevant to the regulation of apo AI gene expression and therefore to plasma high density lipoprotein cholesterol (HDL-C), we explored the association between this MspI polymorphic site and circulating HDL-C levels in 243 healthy Caucasians. There were 143 aged 18-67 years (60 males and 83 females) and 100 aged 6-12 years (58 males and 42 females). We also compared this association with a known MspI polymorphic site, a G to A transition at -75 bp of the apo AI gene. The rare allele (-) frequency for the polymorphism at +83 bp was 4.1% and 22.1% for the polymorphism at -75 bp. Subjects heterozygous for the loss of the MspI restriction site at +83 bp (genotype: M2+-, n = 20) had higher HDL-C levels than M2+2 subjects (mean +/- SD: 1.73 +/- 0.31 vs. 1.41 +/- 0.39 mmol/l, P < 0.05 for adults; 1.71 +/- 0.33 vs. 1.34 +/- 0.29 mmol/l, P < 0.01 for children). Adults with the G to A substitution at -75 bp also had higher HDL-C levels (1.56 +/- 0.36 mmol/l for AA, 1.53 +/- 0.38 mmol/l for GA, and 1.36 +/- 0.38 mmol/l for GG, P < 0.05); this difference was not observed in the children. The MspI polymorphisms at both sites were in linkage disequilibrium. Their joint effect on the HDL-C levels was also significant and individuals with rare alleles (-) at both sites had the highest HDL-C levels. In an analysis of variance, the MspI polymorphism at +83 bp, and at -75 bp and gender independently accounted for 6.5%, 1.7%, and 5.9%, respectively, of the variance in circulating HDL-C levels when age was controlled as a covariate. We conclude that loss of the MspI site by the C to T (+83 bp) and/or the G to A (+84 bp) transitions is highly associated with increased HDL-C levels. The association appears to be more significant than that of the G to A transition at -75 bp.

C to T and/or G to A transitions are responsible for loss of a MspI restriction site at the 5'-end of the human apolipoprotein AI gene.

We detected the loss of a MspI restriction site by a C to T transition at +83 bp and a G to A transition at +84 bp of the 5'-end non-coding region of the human apolipoprotein AI gene. This base change occurred at the "hot spot" (CCGG) for methylation, which may be important in the regulation of gene expression. The population frequency for the loss of the MspI site is 6.1%.

Chemical cleavage of mismatch: a new look at an established method.

Chemical cleavage of mismatch (CCM), also known as chemical mismatch cleavage (CMC) or the HOT (hydroxylamine/osmium tetroxide) chemical method, has been used for detection of sequence variability with many systems since it was first described. Recently, adaptation to fluorescence-based detection systems has fundamentally changed both the execution and analysis of CCM. This review will outline major advances in the methodology of CCM, from the advent of PCR through fluorescent analysis, and includes applications and modifications of CCM.

Reactivity of potassium permanganate and tetraethylammonium chloride with mismatched bases and a simple mutation detection protocol.

Many mutation detection techniques rely upon recognition of mismatched base pairs in DNA hetero-duplexes. Potassium permanganate in combination with tetraethylammonium chloride (TEAC) is capable of chemically modifying mismatched thymidine residues. The DNA strand can then be cleaved at that point by treatment with piperidine. The reactivity of potassium permanganate (KMnO4) in TEAC toward mismatches was investigated in 29 different mutations, representing 58 mismatched base pairs and 116 mismatched bases. All mismatched thymidine residues were modified by KMnO4/TEAC with the majority of these showing strong reactivity. KMnO4/TEAC was also able to modify many mismatched guanosine and cytidine residues, as well as matched guanosine, cytidine and thymidine residues adjacent to, or nearby, mismatched base pairs. Previous techniques using osmium tetroxide (OsO4) to modify mismatched thymidine residues have been limited by the apparent lack of reactivity of a third of all T/G mismatches. KMnO4/TEAC showed no such phenomenon. In this series, all 29 mutations were detected by KMnO4/TEAC treatment. The latest development of the Single Tube Chemical Cleavage of Mismatch Method detects both thymidine and cytidine mismatches by KMnO4/TEAC and hydroxylamine (NH2OH) in a single tube without a clean-up step in between the two reactions. This technique saves time and material without disrupting the sensitivity and efficiency of either reaction.

HLA-G deletion polymorphism and pre-eclampsia/eclampsia.

OBJECTIVE: To investigate a HLA-G deletion polymorphism in pre-eclamptic pedigrees and the general population. DESIGN: A population association study of HLA-G genotypes from pre-eclamptic/eclamptic patients and control groups. SETTING: Analyses undertaken in the School of Biological Sciences, Macquarie University. Patients were from Royal Women's Hospital, Melbourne, and controls were from Westmead Hospital and Macquarie University, Sydney. SUBJECTS: One hundred and ninety-six individuals, consisting of 29 pre-eclamptic/eclamptic (PE/E) patients, 13 individuals born of a PE/E pregnancy, 46 blood relatives of PE/E patients, 21 husbands of PE/E patients, 25 women normotensive in first pregnancy, 15 husbands of women normotensive in first pregnancy and 47 staff and students of Macquarie University. RESULTS: Genotypic and gene frequencies were not significantly different in the seven groups examined. CONCLUSION: There is no detectable relationship between susceptibility to pre-eclampsia or being born of a pre-eclamptic pregnancy and HLA-G genotype.

A genomewide linkage study of preeclampsia/eclampsia reveals evidence for a candidate region on 4q.

Preeclampsia (PE) and eclampsia (E) are potentially life-threatening conditions that can occur during human pregnancy. Generally considered to be different degrees of severity of the same disease process, the PE/E syndrome is thought to be predominantly genetic in origin, although its exact etiology and genetics are not fully understood. Here we report results of a genomewide linkage search for the gene(s) responsible for susceptibility to PE/E, using 15 informative pedigrees and 90 polymorphic DNA markers from all autosomes. Because of uncertainties concerning inheritance and diagnosis, four different models that assume maternal gene expression have been used to carry out LOD-score analysis. The region between D4S450 and D4S610 (2.8 cM) on the long arm of chromosome 4 was identified as a strong candidate region for a PE/E-susceptibility locus. The maximum multipoint LOD score within this interval was 2.9. Analysis of markers in the region around D4S450 and D4S610 by the affected-pedigree-member method also supported the possibility of a susceptibility locus in this region. However, to verify or exclude definitively linkage to this region, other groups of PE/E pedigrees will be required.

Interaction of linear homologous DNA duplexes via Holliday junction formation.

Interaction of linear homologous DNA duplexes by formation of Holliday junctions was revealed by electrophoresis and confirmed by electron microscopy. The phenomenon was demonstrated using a model of five purified PCR products of different size and sequence. The double-stranded structure of interacting DNA fragments was confirmed using several consecutive purifications, S1-nuclease analysis, and electron microscopy. Formation of Holliday junctions depends on DNA concentration. A thermodynamic equilibrium between duplexes and Holliday junctions was shown. We propose that homologous duplex interaction is initiated by nucleation of several dissociated terminal base pairs of two fragments. This process is followed by branch migration creating a population of Holliday junctions with the branch point at different sites. Finally, Holliday junctions are resolved via branch migration to new or previously existing duplexes. The phenomenon is a new property of DNA. This type of DNA-DNA interaction may contribute to the process of Holliday junction formation in vivo controlled by DNA conformation and DNA-protein interactions. It is of practical significance for optimization of different PCR-based methods of gene analysis, especially those involving heteroduplex formation.