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PubMed1996

'Fingerprinting' of HLA-DQA by polymerase chain reaction and heteroduplex analysis.

We have developed a rapid, non-radioisotopic PCR fingerprinting technique for analysis of the HLA-Class II DQA gene second exon polymorphism, and have applied it to DNA samples from 210 healthy individuals. The technique is based on the formation of specific patterns (fingerprints) of homoduplexes or heteroduplexes between in vitro amplified DNA sequences. After electrophoresis on non-denaturing polyacrylamide gels and ethidium bromide fluorescence or silver staining, different HLA-DQA types give allele-specific banding patterns. HLA DQA typing is done by visual comparison between the sample's fingerprint patterns and appropriate controls. Similar fingerprints can be resolved by mixing the sample with a standard DNA in an amplified 'DNA crossmatch'. This application of PCR fingerprinting is useful to confirm the HLA-DQA serological typing and to improve the molecular characterization of this polymorphic region.

Alleles↗
PubMed1994

Detection of two point mutations causing familial defective apolipoprotein B-100 by heteroduplex analysis.

Familial defective apolipoprotein B-100 (FDB) is a dominantly-inherited genetic disorder causing primary hypercholesterolemia and premature coronary heart disease. To date, only two mutations causing FDB have been identified. A rapid non-radioactive technique is described to detect both disease-related apolipoprotein B point mutations in polymerase chain reaction (PCR) products amplified from genomic DNA. Heteroduplex formation between different alleles from FDB heterozygotes was shown to be visible directly after electrophoresis of PCR products and staining in low cross-linking polyacrylamide gels. We found that the simplicity of the method, in addition to its potential to identify new mutations in the amplified PCR product, makes heteroduplex detection the preferred initial method of screening potential heterozygotes.

Apolipoprotein B-100↗
PubMed1976

Isolation and characterization of lambdadargECBH transducing phages and heteroduplex analysis of the argECBH cluster.

Transducing lambda bacteriophages have been isolated which carry the divergently transcribed argECBH operon of E. coli K12 and various portions of the adjacent ppc and bfe chromosomal regions. They were recovered from lysates prepared by the procedure of Schrenk and Weisberg using a Ppc+ Arg+ Bfe+ strain carrying a deletion of the usual attachment site of lambda. Heteroduplex DNA mapping of these lumbdadarg and of the phi 80 darg isolated by B. Konrad indicates that the two kinds of phages carry the arg cluster in opposite orientations, a situation favorable for the isolation of argECBH DNA. A physical map of the ppc argECBH bfe region including 2 unusual attachment sites of lambda has been constructed. The localization of the end points of certain arg deletions provides a useful reference framework for the currently pursued mapping of mutations affecting the control of divergent transcription and for the location of restriction enzyme cleavage sites in the arg region.

Arginine↗
PubMed1984

Heteroduplex analysis of molecular clones of the pathogenic Friend virus complex: Friend murine leukemia virus, Friend mink cell focus-forming virus, and the polycythemia- and anemia-inducing strains of Friend spleen focus-forming virus.

The pathogenic Friend virus complex is of considerable interest in that, although members of this group are genetically related, they differ markedly in biochemical and biological properties. Heteroduplex mapping of molecular clones of the Friend virus complex, which includes the replication-competent ecotropic Friend murine leukemia virus (F-MuLV) and mink cell focus-forming virus (F-MCF) and replication-defective polycythemia- and anemia-inducing strains of spleen focus-forming virus (SFFVp and SFFVa, respectively), was employed to provide insight into the molecular basis of their relationships. In heteroduplexes of F-MuLV X F-MCF, a major substitution of 0.89 kilobases in the env gene of F-MCF was discerned. Heteroduplexes of SFFVp X F-MuLV or F-MCF and SFFVa X F-MuLV or F-MCF showed several major deletions in the pol gene region and a single major deletion in the 3' half of the env gene region of SFFVp and SFFVa. A major substitution of 0.89 kilobases was mapped to the 5' end of the env deletion of SFFVp and SFFVa in heteroduplexes with F-MuLV, similar to that seen in F-MuLV X F-MCF heteroduplexes. In contrast, this env gene region was totally homologous in F-MCF X SFFVp or SFFVa and SFFVp X SFFVa heteroduplexes. Our results suggest that (i) both SFFVp and SFFVa lack part of the env gene at its 3' end, corresponding to the p15(E) coding region, (ii) major deletions occur in the pol and env genes which account for the replication defectiveness of SFFVp and SFFVa, (iii) minor substitutions occur in the gag gene region of SFFVa that are not present in SFFVp, F-MuLV, or F-MCF, (iv) a major substitution exists in the gp70 region of the env gene between F-MuLV and F-MCF that probably accounts for the differences in their host range specificities, (v) this substitution in F-MCF is identical to the gp70 part of the gp52 coding region of SFFVp and SFFVa, and (vi) heteroduplexes to F-MCF show unambiguously that no additional large substitutions are present in SFFVp or SFFVa that could account for differences in their leukemogenicity.

Animals↗
PubMed1984

Comparison of myeloproliferative sarcoma virus with Moloney murine sarcoma virus variants by nucleotide sequencing and heteroduplex analysis.

The myeloproliferative sarcoma virus (MPSV) was derived by passage of Moloney sarcoma virus (Mo-MuSV) in adult mice. Mo-MuSV variants transform fibroblasts. However, MPSV also affects erythroid, myeloid, and hematopoietic stem cells. The MPSV proviral genome, two temperature-sensitive mutants derived from it, Mo-MuSV variant M1, and Moloney murine leukemia virus (Mo-MuLV) were compared by heteroduplex mapping. MPSV wild type was found to have 1 kilobase pair deleted from the pol gene and to contain v-mos-related sequences. The 3' end of MPSV, including the oncogene-helper junctions, the v-mos gene, and the 3' long terminal repeat, was sequenced and compared with sequences of Mo-MuLV, MSV-124, and the mouse oncogene c-mos. From these data, MPSV appears to be either closely related to the original Mo-MuSV or an independent recombinant of Mo-MuLV and c-mos. Five possible explanations of the altered specificity of MPSV are considered. (i) The MPSV mos protein has properties inherent in c-mos but lost by other Mo-MuSV mos proteins. (ii) The MPSV mos protein has altered characteristics due to amino acid changes. (iii) Due to a frameshift, MPSV codes for a mos protein truncated at the amino terminal and also a novel peptide. (iv) A second novel peptide may be encoded from the 3' env region. (v) MPSV has long terminal repeats and an enhancer sequence more like Mo-MuLV than Mo-MuSV, with a consequently altered target cell specificity.

Animals↗
PubMed1997

Rapid molecular haplotyping of the first exon of the human dopamine D4 receptor gene by heteroduplex analysis.

The dopamine D4 receptor gene (DRD4) and its products are of great interest in many neurospsychiatric disorders. There are at least five expressed polymorphisms in exons 1 and 3, plus rare expressed variants, all of which may have functional relevance. Several studies have described methods for studying the exon 3 polymorphisms, especially the VNTR; fewer reports have documented the exon 1 polymorphisms and variants of DRD4. We report here a simple, rapid, nonisotopic, nondenaturing heteroduplex method for determining the molecular haplotype composed of the two more polymorphic systems of the first exon of DRD4: the 12 bp duplication and 13 bp deletion. This method will facilitate future research on expressed variation of this gene.

Electrophoresis, Polyacrylamide Gel↗
PubMed1995

Application of heteroduplex analysis for detecting variation within the growth hormone 2 gene in Salmo trutta L. (brown trout).

A new method to detect variation at a single copy nuclear gene in brown trout, Salmo trutta L., is provided. The technique entails (i) selective gene amplification by the polymerase chain reaction (PCR), (ii) digestion of amplification products by restriction endonucleases to obtain fragments of suitable size, (iii) hybridization with heterologous DNA followed by denaturation and reannealing to obtain heteroduplex molecules, and (iv) screening for variation in polyacrylamide gels. Variation was studied within a growth hormone 2 gene 1489 bp segment and polymorphism was detected in two HinfI-digested fragments. Formation of different heteroduplex patterns in experimental mixtures of digested amplification products from brown trout and Atlantic salmon, Salmo salar L., allowed us to determine the genotype of the brown trout. Polymorphism was observed in four out of six studied populations.

Animals↗
PubMed1976

Fusion of two F-prime factors in Escherichia coli studied by electron microscope heteroduplex analysis.

A fused F prime factor was obtained from a mating of a recA donor carrying an F'- factor containing the genes metBJF, ppc and argECBH (KLF5) with a recA recipient carrying an F' factor containing att80, trp and lac (f155). lysogenization of this fused F-prime factor with gammacI857hphi80 phage followed by thermoinduction produced the transducing phages phi80 dmetBJF and phi80 dppcargECBH. This kind of fusion provides a general procedure for the construction of transducing phages carrying genes from different regions of the E. coli genome. To understand the mechanism of this fusion, the parental F prime factors (F155 and KLF5) were analyzed by the electron microscope heteroduplex technique. F155 has a length of 176 +/- 3 kilobases including two substitutions. The F sequence 0 F-2.8 F has been substituted by 53 kb of chromosomal DNA including the lac operon and the F sequences 8.5 F-16.3 F has been substituted by 27 kb of a chromosome sequence including att80 and the trp operon KLF5 contains 221 +/- 4 kilobases of DNA (molecular weight, 148 megadaltons). It contains complete F and the segment of the E. coli chromosome from polA to rif. The F sequence 2.8 F-8.5 F known to be involved in F specific recombination in recA+ and recA backgrounds occurs twice on KLF5, once at each of the junctions of F DNA with chromosomal DNA. The population of closed circular plasmid molecules extracted from KLF5-containing strains is heterogeneous. It is proposed that this heterogeneity is due to intramolecular recombination events occurring in KLF5 between the duplicated 2.8 F-8.5 F sequences. Such recombination can account for the genetic instability of KLF5 observed in both recA+ and recA hosts. The F sequence 2.8 F-8.5 (also called gammadelta) is one of the characterized integration sequences on F. A model for the fusion of the parental F prime factors is proposed in which recombination between gammadelta sequences brings att80 close to the metBJF genes. This is followed by a deletion of an F' lac factor. The resulting fused F' factor still carries two gammadelta sequences and is therefore expected to be unstable. The closed circular molecules isolated from the fused F' containing strains show two different sizes of molecules. Genetic and physical analyses of these molecules are in agreement with the predicted instability of the fused F' factor and the existance of the gammadelta sequence in the phi80 dmet phages isolated from fused F' and previously analyzed by the electron microscope heteroduplex technique.

Coliphages↗
PubMed2000

Use of haploid mixtures and heteroduplex analysis enhance polymorphisms revealed by denaturing gradient gel electrophoresis.

PCR-based codominant genetic markers were developed by using primer sequences designed from cDNA clones of loblolly pine (Pinus taeda L.). Such markers offer certain advantages relative to simple-sequence repeat (SSR), also known as short-tandem repeat (STR) markers, and include the ability to quantify and map DNA polymorphisms in expressed genes. However, detecting these DNA polymorphisms is more problematic because many DNA polymorphisms in genes involve base substitutions rather than insertions or deletions. Denaturing gradient gel electrophoresis (DGGE) is a sensitive and efficient method for detecting sequence differences among PCR fragments. This paper demonstrates the application of DGGE to genetically map expressed genes in loblolly pine. Also, heteroduplex DNA fragments, formed during the amplification of DNA from heterozygotes and from mixes of haploid DNAs from megagametophytes, enhanced and strengthened genetic interpretations and genotypic classifications.

Alleles↗
PubMed1996

Tracking H-2 alleles in transgenic mice by RFLP and heteroduplex analysis.

Transgenic mice provide valuable tools for biological research including many areas of immunology. In studies involving the major histocompatibility complex (MHC), it is often necessary to place the desired transgene in a specific H-2 (the murine MHC) environment. In this regard, the strains commonly used for the production of transgenic mice also carry well characterized H-2 alleles and provide an appropriate genetic background for MHC related experiments. In this study, a highly polymorphic microsatellite of tetranucleotide repeats from the second intron of the class II Eb gene within the H-2 complex was used in order to identify the corresponding alleles. The relevant H-2 allele(s) along with the transgene were then tracked throughout the production of a chicken ovalbumin-specific transgenic strain. The technique involved PCR-amplification of a DNA sequence encompassing the H-2 specific microsatellite followed by RFLP and heteroduplex analyses. This approach is likely to find wide application in the background checking of transgenic mice, especially in immunological research requiring a defined H-2 background.

Alleles↗