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PubMed1991

The molecular genetic analysis of gene rearrangements in acute lymphoblastic leukaemia.

In ALL the majority of cases possess clonal rearrangements of the Ig or TCR gene loci. Detection of these clonal markers by Southern blot analysis over a disease course has provided information on the fate and origin of leukaemic clones during treatment. Detection of these gene rearrangements has been used to detect residual disease during treatment. More recently, methods have been developed for the detection of Ig and TCR gene rearrangements using the polymerase chain reaction (PCR). This amplification technique allows for the rapid detection of gene rearrangements with a greater sensitivity than more conventional methods. The full impact and usefulness of this technique in residual disease detection has yet to be determined. The presence of the Philadelphia chromosome t(9;22) in ALL is associated with poor prognosis. Its detection by Southern blot is technically complicated due to the heterogeneity of chromosome breakpoints involved. The development of PCR-based methods for the detection of the bcr/abl mRNA associated with the Philadelphia chromosome has improved our understanding of the significance and incidence of this disease marker in ALL, emphasizing the importance of establishing Philadelphia status on all patients at diagnosis. Although longitudinal studies in CML have shown the presence of bcr/abl mRNA to be associated with residual disease, and its absence associated with long-term remission, these studies have yet to be reported for ALL. The usefulness of detection of residual disease using bcr/abl has yet to be determined.

Blotting, Southern↗
PubMed1992

Linkage analysis of spinal muscular atrophy.

Linkage data between four markers on chromosome 5 confirm and extend our previous studies that localized the mutation in spinal muscular atrophy to 5q11.2-q13.3. Localization of D5S6 by in situ hybridization refines the mapping of the defective gene to the region 5q12.2-q13. We also report the use of a highly informative PCR-based polymorphism with five alleles. This RFLP will be particularly useful for prenatal diagnosis where only old tissue samples from affected individuals are available. The high heterozygosity of this locus should also assist in identifying recombinants that will refine the genetic mapping of the mutation.

Chromosome Mapping↗
PubMed1991

SstII polymorphic sites in the promoter region of the human cystatin C gene.

By direct sequencing of polymerase chain reaction (PCR) amplified DNA from different individuals, three points mutations have been found in a 220-bp fragment from the promoter region of the human cystatin C gene. The three mutations are all localized within a short segment of 85bp on the same allele. One of the base substitutions results in the generation of a novel SstII restriction site and another in the loss of the commonly occurring SstII restriction site. A PCR-based assay for analysis of the two SstII sites was designed and used to demonstrate Mendelian inheritance of the polymorphism. This SstII restriction fragment lenght polymorphism offers a new probe-independent marker for chromosome 20 linkage studies.

Base Sequence↗
PubMed1992

Detection of genetic variation between and within populations of Gliricidia sepium and G. maculata using RAPD markers.

Gliricidia sepium and G. maculata are multi-purpose leguminous trees native to Central America and Mexico. Research programmes have been initiated to define the native distribution of Gliricidia and sample the spectrum of genetic variation. To date, there has been little systematic assessment of genetic variability in multi-purpose tree species. Accurate estimates of diversity between- and within-populations are considered a prerequisite for the optimization of sampling and breeding strategies. We have used a PCR-based polymorphic assay procedure (RAPDs) to monitor genetic variability in Gliricidia. Extensive genetic variability was detected between species and the variability was partitioned into between- and within-population components. On average, most (60 per cent) of the variation occurs between G. sepium populations but oligonucleotide primers differed in their capacity to detect variability between and within populations. Population-specific genetic markers were identified. RAPDs provide a cost-effective method for the precise and routine evaluation of variability and may be used to identify areas of maximum diversity. The approaches outlined have general applicability to a range of organisms and are discussed in relation to the exploitation of multi-purpose tree species of the tropics.

Americas↗
PubMed1995

A CD10-positive subset of malignant cells is identified in multiple myeloma using PCR with patient-specific immunoglobulin gene primers.

Immunophenotypic studies show the presence of CD10-bearing malignant cells in a small subset of multiple myeloma (MM) patients. We used a sensitive PCR-based technique in order to determine the frequency that MM patients contain a malignant subpopulation which expresses this antigen. The immunoglobulin (Ig) heavy chain variable region (VH) gene sequence expressed by the malignant clone in MM can be used as a tumor specific marker. After determining this sequence in six MM patients, patient specific VH oligonucleotide primers from complementarity determining region (CDR) sequences were generated. Bone marrow mononuclear cells from these patients were incubated with two different anti-CD10 antibodies or isotype identical murine IgG controls. Cells were then sorted by flow cytometry into the 1% brightest cells containing > 99.99% CD10-positive cells and two fractions including the 90 and 10% dimmest staining cells. PCR amplification was performed on DNA from approximately 10(4) cells (0.1 microgram) using patient specific CDR1 and CDR3 primers. Detectable PCR product was obtained in each sorted sample although the intensity of the band was much higher in cells lacking CD10 expression (the 90 and 10% dimmest fractions) than in the CD10-bearing (1% brightest) population. These results imply that there is a small population of CD10-bearing clonal cells in most, if not all patients with MM.

Base Sequence↗
PubMed1992

Amplification of three hypervariable DNA regions by polymerase chain reaction for paternity determinations: comparison with conventional methods and DNA fingerprinting.

The present study evaluates the usefulness of a PCR-based method for routine paternity testing in 35 paternity cases. This identification method which is based on amplification of three hypervariable genetic loci, apoB, D1S80 and HLA-DQ alpha, is compared, with regard to reliability and technical feasibility, to the conventional identification methods based on protein polymorphisms and to Southern blot hybridizations with multi- and single locus probes. Data obtained by PCR-amplification of these three loci resulted in paternity indices (56.1, geometric mean value) which are at the same level as the corresponding values derived from standard genetic blood group markers (42.7). The geometric mean value of the paternity indices obtained by Southern blot hybridization using three single locus probes (190.6) was more informative, and the most informative analysis proved to be Southern blot hybridization with multilocus probes. The technical feasibility and the reproducibility of the PCR-based analysis is, however, overwhelming, and if several highly polymorphic loci are amplified, the resolving power of PCR-analysis is similar to that obtained using multilocus probes.

Blood Grouping and Crossmatching↗
PubMed1995

Detection of loss of heterozygosity in the p53 tumor suppressor gene using a PCR-based assay.

Inactivation of the p53 tumor suppressor gene has been reported to be a prognostic factor in several human cancer types. Normal function of the gene is affected by deletion in one allele; dysfunction of the other allele is often caused by a mutation. In tumors of heterozygous individuals, deletion of one allele can be detected as loss of heterozygosity (LOH). A recently found variable number of tandem repeats (VNTR) segment in intron 1 of the p53 gene seems to be highly polymorphic and, therefore, a very useful marker in detecting LOH in various types of tumor samples. We in vitro amplified the VNTR segment from genomic DNA samples of 101 lung cancer patients and run conventional agarose gel electrophoreses in order to detect the alleles of various length, differing by the number of repeats. The usefulness of the method was studied using DNA from white blood cell samples and from fresh and formalin-fixed, paraffin-embedded tumor samples. Of the patients, 56% were found to have two different alleles, i.e. were informative in this assay. In 18% of the lung tumors from the informative cases, LOH in the p53 suppressor gene was detected.

Adenocarcinoma↗
PubMed1991

Nonradioactive assay for new microsatellite polymorphisms at the 5' end of the dystrophin gene, and estimation of intragenic recombination.

Indirect tracking of mutation by DNA polymorphisms is still essential for carrier and prenatal diagnosis of Duchenne/Becker muscular dystrophy, at least in the families where no deletion can be detected. Because of the relatively high level of intragenic recombination, informative and easily testable markers at both ends of the gene are necessary for efficient and accurate diagnosis. We report the characterization of two polymorphic microsatellite sequences (TG repeats) at the 5' end of the dystrophin gene, within 40 kb of the muscle-specific promoter. The most useful one (5' DYS MSA) has 10 alleles with a 57% heterozygosity and can be tested on small polyacrylamide gels in a nonradioactive PCR-based assay. Despite its large number of alleles, this microsatellite shows strong linkage disequilibrium with a two-allele polymorphism reported by Roberts et al., an indication of the stability of this type of sequences. We have used the new microsatellites at the 5' end, along with one we reported previously for the 3' end, to type the families in the CEPH (Centre d'Etude du Polymorphisme Humain) panel. While the number of informative families has increased by a factor of about two with respect to the study of Abbs et al., the estimates of the recombination fractions are in good agreement with this previous report, suggesting a 11% recombination across the gene (3% between the 5' end and the pERT87 region, 8% between pERT87 and the 3' end), which is about fivefold more than expected. However, these estimates still have wide confidence limits.

Alleles↗
PubMedDate not supplied

A rapid PCR-based method to distinguish between fetal and maternal cells in chorionic biopsies using microsatellite polymorphisms.

In some chorionic villus biopsy (CVB) cases the fetal/maternal origin of the tissue obtained is uncertain. An approach which only requires small amounts of CVB tissue to establish its origin is described. Since it is only the samples typed as female that could be either fetal or maternal, a paternal X chromosome contribution is sought by using highly polymorphic X-linked microsatellites.

Alleles↗
PubMed1992

Leishmaniasis epidemiology: all down to the DNA.

Application of quantitative methods to the study of leishmaniasis epidemiology has allowed Dye (1992) to pinpoint important biological parameters which, if they could be accurately measured in the field, would contribute most to our knowledge of the spread of disease and key targets for control. Three areas in which laboratory-based research could impact most on leishmaniasis epidemiology were highlighted by Dye (1992): (i) the development of accurate diagnostic tools which can distinguish between current and past infection; (ii) to determine the underlying molecular/genetic basis to virulence polymorphisms in the parasite and study these in the context of field epidemiological studies; and (iii) to provide the molecular tools to measure genetic variation in resistance to infection in humans and in reservoir hosts of disease. This paper describes current progress in attaining these goals, highlighting first the work on isolation and field application of genomic and kDNA probes for species-specific diagnosis, and the development of PCR-based assays which can be performed under field conditions. At a more preliminary stage, studies are described in which variability in the major molecular determinants of virulence (lipophosphoglycan, GP63, and members of the HSP70 family of stress proteins) identified through studies of laboratory models of infection, is being measured in primary field isolates of Leishmania peruviana. To complete the picture, current progress in identifying and cloning the genes which control host resistance to leishmanial infection is described, along with field studies of multicase families of human disease in which linkage analysis using marker genes from the chromosomal regions bearing these genes can be used to find evidence for their role in determining disease phenotypes in man. The projected view from these studies is that the future of leishmaniasis epidemiology will be all down to the DNA.

Animals↗
PubMed1992

Polymerase chain reaction-based genotyping for allotypic markers of immunoglobulin kappa shows allelic association of Km with kappa variable segment.

Allotypic markers of immunoglobulin kappa (Km) may be determined using a novel method of amplification of the constant segment (C kappa) (IGKC) by polymerase chain reaction (PCR) followed by restriction enzyme digestion. Restriction sites in the C kappa PCR product correlate with allotypic differences among Km(1), Km(1,2), and Km(3) alleles. An AccI site in the PCR product correlates with Km(3); and presence or absence of a MaeII site correlates with the Km(1) or Km(1,2) allele, respectively. Km allelic frequencies were determined in a Caucasian population and compared to genotypic frequencies of nearby polymorphic markers. Among unrelated individuals with rheumatoid arthritis (RA) and controls, there is no evidence of allelic association between CD8A and polymorphic markers of the immunoglobulin kappa region [a V kappa (IGKV) BglII polymorphism about 24 kb centromeric to C kappa, Km allotype, and a SacI polymorphism 3.5 kb telomeric to the C kappa segment]. Similarly, there is no allelic association of the SacI C kappa polymorphism with Km or with the BglII V kappa polymorphism. However, there is evidence of allelic association of V kappa B3 and Km, specifically between the V kappa BglII 2.2-kb allele and Km(3) and also between the V kappa 3.5-kb allele and Km(1,2). Therefore, Km typing by PCR-based methods suggests the presence of allelic association between polymorphisms within the coding region of the C kappa segment and the nearest V kappa segment.

Alleles↗
PubMed1992

A PCR-based method of identifying species-specific repeated DNAs.

A PCR-based method is described to facilitate the identification of DNA fragments that are highly repeated and species-specific. The precision of the technique was demonstrated by cloning a fragment that occurred in a high number of copies in the plant species Medicago granadensis but in a low number of copies in 17 other Medicago species and Melilotus officinalis. This method should greatly accelerate the isolation and cloning of short and long interspersed nuclear elements with species-specific distributions. Such clones should prove useful in studies of phylogenetic relationships in the identification of interspecific hybrids, as in situ chromosome markers and other applications.

Base Sequence↗
PubMed1992

Genetic and physical mapping on chromosome 4 narrows the localization of the gene for facioscapulohumeral muscular dystrophy (FSHD).

We have used a combination of classical RFLPs and PCR-based polymorphisms including CA repeats and single-strand conformation polymorphisms to generate a fine-structure genetic map of the distal long arm of chromosome 4q. This map is now genetically linked to the pre-existing anchor map of 4pter-4q31 and generates, for the first time, a complete linkage map of this chromosome. The map consists of 32 anchor loci placed with odds of greater than 1,000:1. The high-resolution map in the cytogenetic region surrounding 4q35 provides the order 4cen-D4S171-F11-D4S187-D4S163-D4S139-4q ter. When we used somatic cell hybrids from a t(X;4)(p21;q35) translocation, these five markers fell into three groups consistent with the genetic map-D4S171 and F11 in 4pter-4q35, D4S163 and D4S139 in 4q35-4qter, and D4S187 as a junction fragment between these two regions. These markers are in tight linkage to the gene for facioscapulo-humeral muscular dystrophy (FSHD) mapped to this region by several collaborating investigators and provide a framework for further detailed analysis of this region.

Base Sequence↗
PubMed1991

Differentiation of Fusarium solani f. sp. cucurbitae races 1 and 2 by random amplification of polymorphic DNA.

We have used a PCR-based technique, involving the random amplification of polymorphic DNA (RAPD), to assess genome variability between 21 isolates from F. solani f. sp. cucurbitae races 1 and 2. Based on RAPD marker patterns the isolates fell into two distinct groups corresponding to mating populations MPI and MPV. Four isolates that could not be assigned to one or other mating population by traditional means were distinguished by RAPD patterns. Seven polymorphic RAPD products were used to probe Southern blots of MPI and MPV genomic DNA. Six of the seven probes hybridized to single-copy sequences and five of the seven probes showed specificity for one or other mating population. We suggest that not only is the technique a rapid and reliable tool for isolate-typing of fungi but it also provides a rapid method for obtaining species- or race-specific hybridization probes.

Base Sequence↗
PubMed1991

The polymerase chain reaction: a new tool for the detection of minimal residual disease in haematological malignancies.

The polymerase chain reaction (PCR) is a novel technique for the in vitro amplification of specific short DNA fragments, which permits a selective and up to 10(7) fold enrichment of the target sequence. The method is increasingly being used for the molecular genetic analysis of hereditary, infectious and neoplastic disorders. The use of PCR for the detection of minimal residual disease in particular types of leukaemia or lymphoma, such as chronic myelogenous leukaemia expressing specific BCR/ABL-RNA and follicular non-Hodgkin lymphoma with the chromosomal translocation t(14;18) are reviewed. In acute lymphoblastic leukaemia clone-specific sequences from rearranged antigen receptor genes may be molecular markers suitable for amplification. Although PCR holds great promise for "molecular" staging and follow-up, several technical problems have to be kept in mind, and the clinical relevance of PCR-based evidence of minimal residual disease in haematological malignancies requires further investigation.

False Negative Reactions↗
PubMed1991

Characterization and rapid analysis of the highly polymorphic VNTR locus D4S125 (YNZ32), closely linked to the Huntington disease gene.

The highly polymorphic VNTR locus pYNZ32 has been more extensively characterized, and its analysis converted to a rapid PCR-based format. DNA sequencing in the areas within and flanking the repeated segment allowed the design of specific amplification primers. The repeated region of pYNZ32 consists of an imperfectly duplicated 27-bp motif, 16 bases of which are more highly conserved. Allelic products from PCR amplification were resolved into nine different size classes ranging from approximately 1400 to 2200 bp. Additional polymorphism was revealed when the amplified products were analyzed by restriction enzyme digestion. Both the overall size variation and the internal sequence polymorphism were used to determine a heterozygosity value of 86% for YNZ32 in 50 unrelated individuals. The rapid analysis and improved resolution of amplified alleles on agarose gels, and the internal variability within YNZ32, increase its diagnostic utility as a VNTR and as a linkage marker for the nearby Huntington disease gene.

Base Sequence↗
PubMed1992

PCR detection and differentiation of Chlamydia pneumoniae, Chlamydia psittaci and Chlamydia trachomatis.

A PCR-based system was developed for the detection and differentiation of Chlamydia trachomatis, Chlamydia psittaci and Chlamydia pneumoniae. A conserved 145 bp fragment of the chlamydial omp1 gene was amplified from all three species. The three species were then differentiated from each other by digestion of this PCR product with restriction enzymes Eco RI and either Hind III or Pst I. The system was shown to work for two strains of C. pneumoniae, 11 strains of C. psittaci and 10 serovars of C. trachomatis, and had a sensitivity of less than 10 chlamydial elementary bodies. This method was also applicable to the detection of C. trachomatis in conjunctival and nasopharyngeal swabs.

Adult↗
PubMed1992

A method for specific amplification and PCR sequencing of individual members of multigene families: application to the study of steroid 21-hydroxylase deficiency.

Mutations at the human HLA-linked CYP21B locus are responsible for 21-hydroxylase deficiency, a recessively inherited disorder of steroidogenesis. The scope for PCR-based analysis of the CYP21B gene has been restricted by the very high sequence homology between CYP21B and a closely related pseudogene, CYP21A. Here we describe a novel PCR sequencing strategy that allows the independent amplification of the entire CYP21B coding sequence and the subsequent enzyme-mediated conversion of the PCR product to a single-stranded form for dideoxy sequencing. We have used this approach to characterize the 21-hydroxylase deficiency allele associated with HLA-B55, the most frequent HLA marker associated with a CYP21B point mutation in the British population, and also an HLA-B35 associated allele of Asian origin. Allele-specific oligonucleotide (ASO) hybridization analyses have confirmed the selective amplification of CYP21B genes and the identity of the pathological mutations. The method can be adapted to permit selective amplification and PCR sequencing of individual closely related members of other multigene families and small-copy-number repetitive DNA families.

Adrenal Hyperplasia, Congenital↗