Biomedical subjects

D G Monckton

Publications and source records attributed to D G Monckton.

27 records · Page 2Linked to original sources

PubMed1994

Complex gene conversion events in germline mutation at human minisatellites.

Mutation at the human minisatellites MS32, MS205 and MS31A has been investigated by characterizing mutant alleles in pedigrees and in the case of MS32 by direct analysis of mutant molecules in single sperm. Most mutations at all three loci are polar, involving the preferential gain of a few repeat units at one end of the tandem repeat array. Incoming repeats can be derived from the same allele or the homologous chromosome, through they are frequently rearranged during mutation. Lack of exchange of flanking markers suggests the involvement of complex conversion-like events in the generation of mutant alleles. At MS32, high frequency mutation processes in sperm appear to be largely germline specific and to occur at a constant rate irrespective of allele size. Together with mutational polarity, this implies that germline instability is controlled by elements outside the tandem repeat array.

Alleles↗

PubMed1994

Minisatellite mutation rate variation associated with a flanking DNA sequence polymorphism.

Human minisatellite mutation in the male germline frequently involves complex interallelic gene conversion events restricted to one end of the tandem repeat array. Some alleles at minisatellite MS32 show reduced variability in human populations and are associated with a G to C transversion upstream of the array. Analysis of single sperm demonstrated a frequently profound reduction in mutation rate at alleles carrying the C variant. This mutation suppression acts in cis, but does not affect the ability of an allele to act as sequence donor during gene conversion. This mutation rate polymorphism provides strong evidence for elements near the minisatellite that regulate tandem repeat instability.

Africa↗

PubMed1993

Minisatellite variant repeat mapping: application to DNA typing and mutation analysis.

Most DNA typing systems assay allele length variation at tandemly repeated loci such as minisatellites and microsatellites. Allele length measurements are approximate, which impedes the use of such loci in forensic analysis and in studies of allelic variability at hypervariable loci. We now review progress in the development of alternative DNA typing systems based on allelic variation in the interspersion patterns of variant repeat units along minisatellite alleles. Minisatellite variant repeat mapping by PCR (MVR-PCR) not only provides a powerful new digital approach to DNA typing, but also for the first time allows investigation of the true level of allelic variability at minisatellite loci and of the mutational mechanisms that generate ultravariability.

Alleles↗

PubMed1993

DNA profiling.

Although some concerns still remain in standard DNA profiling technology over the assumptions from population genetics used to calculate expected match frequencies, forensic scientists are preparing for the introduction of the next generation of DNA profiling techniques based on the polymerase chain reaction. These new techniques offer the prospect of dramatically increasing the speed and sensitivity of DNA profiling and have already been applied in some casework studies.

Base Sequence↗

PubMed1993

Four-state MVR-PCR: increased discrimination of digital DNA typing by simultaneous analysis of two polymorphic sites within minisatellite variant repeats at D1S8.

Minisatellite variant repeat mapping by PCR (MVR-PCR) provides a digital approach to DNA typing that can reveal huge levels of variation at minisatellite loci. MVR-PCR has so far been applied to three human minisatellites, including the hypervariable locus D1S8. Previous analysis at D1S8 was based on the discrimination of repeat unit types that differ by a single base substitution. We now show that a second polymorphic site within D1S8 repeats may be assayed simultaneously with the first to define four classes of repeat units ('four-state MVR-PCR'). This approach can also be applied to the other end of D1S8 alleles in 'reverse four-state MVR-PCR'. Both of these procedures substantially increase the informativeness of MVR analysis at D1S8 and should prove useful in studies of minisatellite biology and potentially in forensic DNA typing.

Alleles↗

PubMed1993

Allele-specific MVR-PCR analysis at minisatellite D1S8.

Minisatellite variant repeat mapping by the polymerase chain reaction (MVR-PCR) provides a digital approach to DNA typing of great potential use both in forensic medicine and, by mapping single alleles, for exploring allelic variability and mutation processes at minisatellites. The MVR haplotypes of single alleles can be determined either from physically separated alleles or by pedigree analysis of digital diploid codes generated from both alleles simultaneously. We now show that single alleles can be rapidly mapped from total genomic DNA using allele-specific PCR primers directed to polymorphic sites in the DNA flanking the minisatellite. This approach can also be used to dissect mixed DNA samples such as those often encountered in forensic DNA analysis.

Alleles↗

PubMed1992

Minisatellite variant repeat (MVR) mapping: analysis of 'null' repeat units at D1S8.

Minisatellite variant repeat mapping by PCR (MVR-PCR) is a new approach to studying variation in human DNA which analyses interspersion patterns of variant repeats within minisatellite arrays. MVR-PCR has been applied to the hypervariable human minisatellite D1S8 which contains two major classes of variant 29bp repeat units designated a-type and t-type. The MVR-PCR assay uses a- or t-type specific primers, together with an amplimer at a fixed site in the DNA flanking the minisatellite, to reveal the interspersion patterns of variant repeats along an allele. Extreme levels of variation are seen both in the internal structures of individual alleles and in the digital code generated from the two superimposed alleles in total genomic DNA. However, occasional repeat units fail to amplify in MVR-PCR, signifying the existence of further repeat sequence variants termed 'null' or O-type repeats. Although not significant in individual identification, correct genotyping of null repeats is important when using MVR digital codes in parentage analysis. We have therefore characterised these null repeats and show that most null repeats share a common variant repeat sequence. We discuss the possible origins of null repeats and their application to paternity testing and the analysis of minisatellite evolution.

Alleles↗

PubMed1991

Minisatellite repeat coding as a digital approach to DNA typing.

Most DNA typing systems used in forensic and legal medicine assay allelic length variation at tandem repetitive DNA regions such as minisatellites. A simple alternative approach that displays patterns of variant repeat units along minisatellite alleles is described here. This produces DNA profiles as extraordinarily variable digital sequences appropriate for forensic investigations, including computer databasing, and for analysing allele diversity and the role of recombination in minisatellite instability.

Base Sequence↗

PubMed1991

Minisatellite "isoallele" discrimination in pseudohomozygotes by single molecule PCR and variant repeat mapping.

The D1S8 hypervariable minisatellite MS32 has a heterozygosity of 97.5% based on detectable differences in allele length using standard Southern blot analysis. It has previously been shown that the basic repeat unit is in itself variable and that this may be used to map the internal structure of an allele. This method has already been used to establish that alleles of the same length may have differing internal structures between nonrelated individuals. We now extend this approach to demonstrate that two apparently homozygous individuals are in fact heterozygotes. For each individual the two comigratory alleles were separated, without cloning, using single molecule dilution (SMD) of genomic DNA and recovery with PCR. Mapping of the variant repeat units revealed highly diverged internal structures and, for one individual, a size difference of one repeat unit (29 bp). SMD and PCR recovery provide an efficient system for separating comigratory alleles without prerequirement for knowledge of sequence differences.

Alleles↗