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PubMed2000

Effective capillary electrophoresis-based heteroduplex analysis through optimization of surface coating and polymer networks.

The efficacy of capillary electrophoresis for detecting DNA mutations via heteroduplex analysis (HDA) is dependent upon both the effective passivition of the capillary surface and the choice of the correct polymer network for sieving. Using HDA with laser-induced fluorescence detection of fluorescently labeled DNA fragments, an effective coating and optimal polymer matrix were sought. Optimized separation conditions were determined through the methodological evaluation of a number of different silanizing reagents, polymeric coatings, and polymer networks for resolving the PCR-amplified DNA fragments associated with five mutations (185delAG, 1294del40, 4446C > G, 5382insC, 5677insA) in the breast cancer susceptibility gene (BRCA1). For capillary coating, allyldimethylchlorosilane, 4-chlorobutyldimethylchlorosilane, (gamma-methacryloxypropyl)trimethoxysilane, chlorodimethyloctylsilane (OCT), and 7-octenyltrimethoxysilane were evaluated as silanizing reagents in combination with poly(vinylprrolidone) (PVP) and polyacrylamide (PA) as the polymeric coat. The HDA results were compared with those obtained using a commercial (FC) coated capillary. Of these, the OCT-PVP combination was found to be most effective. Using this modified capillary, HDA with polymer networks that included hydroxyethylcellulose (HEC), linear polyacrylamide, and PVP showed that a PVP-, PA-, or FC-coated capillary, in combination with HEC as the sieving polymer, could be used effectively to discriminate the mutations in less than 10 min. However, optimal performance was observed with the OCT-PVP-coated capillary and HEC as the polymer network.

DNA↗

PubMed1996

Heteroduplex analysis in hemophilia B: detection of two novel factor IX gene mutations.

Heteroduplex analysis of polymerase chain reaction (PCR)-amplified factor IX (FIX) sequences in eight hemophilia B pedigrees localized the causative hemophilia mutation to a single exon in each case. Subsequent PCR-based direct DNA sequence analysis identified two novel FIX mutations and six recurrent mutations. Three of the eight pedigrees represent sporadic hemophilia B, and direct mutation analysis facilitated hemophilia carrier diagnosis in each case.

DNA Mutational Analysis↗

PubMed2005

Seminested polymerase chain reaction and heteroduplex analysis detects the monoclonality of IgH rearrangement in follicular lymphoma patients with high sensitivity.

A new method, combining seminested polymerase chain reaction (PCR) with heteroduplex analysis, was utilized to detect follicular lymphoma (FL) cells in peripheral blood. The method, based on the detection of IgH rearrangements in DNA, detected the presence of monoclonal B cells in FL patients with a high frequency.

B-Lymphocytes↗

PubMed2004

Combined semi-nested polymerase chain reaction and heteroduplex analysis for detecting monoclonality of IgH rearrangement in patients with follicular lymphoma.

A new, sensitive method combining seminested polymerase chain reaction (PCR) and heteroduplex analysis was used to detect follicular lymphoma (FL) cells in peripheral blood. Based on the detection of IgH rearrangement in DNA from peripheral blood leukocytes, the method demonstrated the presence of monoclonal B cells in FL patients with high frequency.

Antibodies, Monoclonal↗

PubMed2000

Analysis of variability of P1 gene region of N strain of potato virus Y using temperature-gradient gel electrophoresis and DNA heteroduplex analysis.

Reaction conditions specific for reverse taranscription-polymerase chain reaction (RT-PCR) of potato virus Y strain NTN (PVYNTN) were used to amplify a 394 bp fragment of the P1 gene from selected PVY isolates with the aim to study the PVY variability within this genomic region. The P1 gene fragment from the Nicola isolate (Nicola P1/1 clone) was sequenced and characterized by temperature-gradient gel electrophoresis (TGGE). The Nicola P1/1 clone differed from that from the Hungarian isolate by double point mutation resulting in two changes at the deduced amino acid level. The clone showed simple transition from double-stranded to single-stranded form with two characteristic melting end points of about 41 degrees C and 48 degrees C. A more complicated TGGE pattern was, however, found for the whole P1 cDNA library of the Nicola isolate, suggesting accumulation of some minor sequence variants of PVY in this isolate. Based on the TGGE pattern, 46 degrees C was selected as the standard temperature for electrophoretic analysis of heteroduplex DNAs formed with the Nicola P1/1 DNA as reference. More than 40 other PVY isolates from PVYN group were analysed using this method. In most cases only minor fractions of electrophoretically distinguishable DNA heteroduplexes were found, however, in most isolates of PVYN-Wilga type, mixtures of the major sequence variants were observed. Two of these variants from the hybrid 220-5 (Czech Republic) were sequenced. Both of them differed from the Nicola P1/1 clone by 6 point mutations, which led to several changes at the amino acid level.

Amino Acid Sequence↗

PubMed2000

Simplified hepatitis C virus genotyping by heteroduplex mobility analysis.

Heteroduplex mobility analysis (HMA) was used to genotype hepatitis C viruses (HCV) with PCR fragments derived from the 5' untranslated region (5'-UTR) or the NS5b region. HCV 5'-UTR fragments were amplified from 296 serum samples by use of a combined reverse transcription-PCR assay, and the genotypes of isolates were determined by sequencing. HCV genotype distributions in Australia were 39% for genotype 1a, 15% for 1b, 3% for 1a/b, <1% for 2a/c, 5% for 2b, 34% for 3a, <1% for 3b, and 1% for 4, and 1% of patients were infected with more than one genotype. Pairwise HMA of subtypes 1a, 1b, 2a/c, 2b, 3a, 3b, 4a, and 6a demonstrated that five distinct heteroduplex patterns were formed between the eight subtypes. A reference panel that contained a representative of each pattern (1a, 2b, 3a, 4a, and 6a) was used for genotyping. The pattern of heteroduplexes formed when a test isolate was mixed with the five reference isolates was correlated with the genotype, as determined by sequencing. Genotypes determined by HMA correlated exactly with sequencing results within the groups 1, 2, 3a, 3b/4, and 6. HMA was also used to simplify the identification of mixed infection with two HCV genotypes. In further studies, with amplicons from the NS5b region, HMA classified isolates into their respective subtypes, and the heteroduplex mobility ratio correlated closely with nucleotide sequence variation at the isolate, subtype, and genotype levels. HMA provides an adaptable, inexpensive, and rapid method of genotyping HCV that requires fewer resources than DNA sequencing.

5' Untranslated Regions↗

PubMed2006

An optimized microchip electrophoresis system for mutation detection by tandem SSCP and heteroduplex analysis for p53 gene exons 5-9.

With the complete sequencing of the human genome, there is a growing need for rapid, highly sensitive genetic mutation detection methods suitable for clinical implementation. DNA-based diagnostics such as single-strand conformational polymorphism (SSCP) and heteroduplex analysis (HA) are commonly used in research laboratories to screen for mutations, but the slab gel electrophoresis (SGE) format is ill-suited for routine clinical use. The translation of these assays from SGE to microfluidic chips offers significant speed, cost, and sensitivity advantages; however, numerous parameters must be optimized to provide highly sensitive mutation detection. Here we present a methodical study of system parameters including polymer matrix, wall coating, analysis temperature, and electric field strengths on the effectiveness of mutation detection by tandem SSCP/HA for DNA samples from exons 5-9 of the p53 gene. The effects of polymer matrix concentration and average molar mass were studied for linear polyacrylamide (LPA) solutions. We determined that a matrix of 8% w/v 600 kDa LPA provides the most reliable SSCP/HA mutation detection on chips. The inclusion of a small amount of the dynamic wall-coating polymer poly-N-hydroxyethylacrylamide in the matrix substantially improves the resolution of SSCP conformers and extends the coating lifetime. We investigated electrophoresis temperatures between 17 and 35 degrees C and found that the lowest temperature accessible on our chip electrophoresis system gives the best condition for high sensitivity of the tandem SSCP/HA method, especially for the SSCP conformers. Finally, the use of electrical fields between 350 and 450 V/cm provided rapid separations (<10 min) with well-resolved DNA peaks for both SSCP and HA.

Acrylic Resins↗

PubMed1999

Single-strand conformation polymorphism and heteroduplex analysis for gel-based mutation detection.

Single-strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) are popular electrophoretic methods for the identification of sequences. The principle reasons for the popularity of these two methods are their technical simplicity and their relatively high sensitivity for the detection of mutations. Here we review the theory and practice of SSCP and HA, including the factors contributing to the sensitivity of mutation detection. For SSCP analysis, these factors include: choice of gel matrix, electrophoretic conditions, presence of neutral additives, fragment size, and G+C content For HA, the principle factors influencing sensitivity are the gel matrix and the identity of the base mismatch.

Acrylic Resins↗

PubMed1992

A new approach to HLA-DPB1 typing combining DNA heteroduplex analysis with allele-specific amplification and enzyme restriction.

Allelic polymorphism of HLA-class II antigens plays a key role in the regulation of the immune response and in transplantation immunity. The allelic diversity of these antigens can now be analyzed at the DNA level after amplification by polymerase chain reaction. In this study we apply a simple technique based on the electrophoretic analysis of DNA heteroduplexes to the typing of HLA-DPB1 alleles. In order to increase its resolution, a group-specific amplification was used which subdivides the 19 HLA-DPB1 alleles in two non-overlapping families. A separate analysis was then performed within each group of alleles. This approach allowed an unequivocal one-step typing of the alleles belonging to group 1 which comprises few alleles of high frequency. Some group 2 alleles require, as a further step, the test with a restriction enzyme. The combination of more than one technique represents, in our opinion, the easiest way to solve the micropolymorphism of class II alleles. We conclude that this method, which is very simple, quick, and accurate and does not require probes, may become the method of choice for HLA-DPB1 typing.

Alleles↗

PubMed1994

A practical approach to HLA-DR genomic typing by heteroduplex analysis and a selective cleavage at position 86.

A common problem facing HLA-typing laboratories is to substitute genomic typing for serology without having to handle a large number of oligoprobes, primers, or restriction enzymes. A protocol is described for HLA-DRB1 genomic typing using a combination of PCR amplification, DNA heteroduplex analysis, and restriction enzymes. Because the core of the procedure is the analysis of the DNA heteroduplexs, it shall be termed HET typing. There are two stages: the first stage comprises two rounds of PCR amplification of the polymorphic second exon of the HLA-DRB genes directly on lysed blood cells. The first amplification is with DRB generic primers, and the second amplification with seven HLA-DRB1 group-specific primers at the 5' end and a common 3' primer. The latter is designed with two nucleotide mismatches, thus creating an artificial restriction site to differentiate between both HLA-DRB1 variants at position 86, which is of critical importance in antigen presentation. The second stage involves subjecting the final amplified product to both DNA heteroduplex formation and digestion by two single-cutter restriction endonucleases. The digested or heteroduplexed samples are run on the same polyacrylamide gel. A total of 25 HLA-DRB1 alleles can thus be differentiated with a total of 10 primers and two restriction enzymes and without the use of probes. This protocol is ideally suited to preliminary HLA class II typing of bone marrow donors.

Alleles↗

PubMed1998

Heteroduplex analysis: a useful screening method for glycogen storage disease type Ia.

Glycogen storage disease type Ia (GSDIa), also known as von Gierke disease, is the most common and severe disease of glycogenoses and is caused by a deficiency of glucose-6-phosphatase (G6Pase) and transmitted by an autosomal recessive trait. The encoding gene of G6Pase is composed of only five exons and each exon is short. With heteroduplex analysis (HDA) method, we analyzed the genomic DNA from a patient diagnosed with GSDIa and from her parents. Exons II and IV of the patient showed heteroduplex bands. The mother had a heteroduplex band of exon II, and the father had a heteroduplex band of exon IV. In a mini-slab electrophoresis, exons II and IV of the patient did not show clear heteroduplex bands, but they appeared broader than the others, which made us suspect that they were heteroduplex bands. HDA is an easy and simple method and can verify mutant homozygous DNA fragments by adding wild-type DNA. We think that HDA may be a very useful screening method for the detection of novel genomic mutation in GSDIa in large-scale and mini-slab electrophoresis.

Adult↗

PubMed2005

Heteroduplex analysis of minisatellite variability.

Minisatellites are tandem repeat arrays of middle size (5-100 bp) repeat units widely distributed in eukaryotic genomes. They have been related to several important features of human genome biology, including gene regulation, chromosomal fragile sites, and imprinting. In this report, we have critically assessed and employed heteroduplex analysis (HA) for the identification of different human minisatellite MsH43 alleles. This minisatellite is organized as a repeat array of 5-6 bp units spanning 0.5 kbp. Our results demonstrate that this procedure is an easy, rapid, and reliable method to document allelic diversity for this locus. This work suggests that HA will also be a useful tool for studying the polymorphism of other minisatellites with small repeat units.

Chromosomes, Human, Pair 2↗

PubMed1998

The combination of heteroduplex analysis and protein truncation test for exact detection of the APC gene mutations.

Familial adenomatous polyposis (FAP) is usually associated with mutation in the adenomatous polyposis coli (APC) gene. To examine the occurrence of these mutations in the number of FAP suspected families from the whole Slovakia effectively, we have applied heteroduplex analysis (HDA) and protein truncation test (PTT) for the analyses of 2-5 base pair deletions and point mutations of the APC gene. In the analyzed exon 15 of the APC gene determined by the primers 15Efor-15Grev for HDA and 15ET7-15J3 for PTT more than 70% of mutations should be deletions [3, 12], which are detectable by HDA. In our collection of 5 FAP families mutations in the APC gene were found in families 10, 27 and 41 using HDA. By PTT test the formation of truncated APC protein in FAP families 2, 10, 16 and 27 were revealed. The necessity of combination of at least HDA and PTT techniques for exact detection of APC mutations in analyzed APC region is discussed.

Adenomatous Polyposis Coli Protein↗

PubMed2003

High-throughput mutation detection method to scan BRCA1 and BRCA2 based on heteroduplex analysis by capillary array electrophoresis.

BACKGROUND: Scanning for mutations in BRCA1 and BRCA2 in a large number of samples is hampered by the large sizes of these genes and the scattering of mutations throughout their coding sequences. Automated capillary electrophoresis has been shown to be a powerful system to detect mutations by either single-strand conformation polymorphism or heteroduplex analysis (HA). METHODS: We investigated the adaptation of gel-based HA of BRCA1 and BRCA2 to a fluorescent multicapillary platform to increase the throughput of this technique. We combined multiplex PCR, three different fluorescent labels, and HA in a 16-capillary DNA sequencer and tested 57 DNA sequence variants (11 insertions/deletions and 46 single-nucleotide changes) of BRCA1 and BRCA2. RESULTS: We detected all 57 DNA changes in a blinded assay, and 2 additional single-nucleotide substitutions (1186 A>G of BRCA1 and 3624 A>G of BRCA2), previously unresolved by conformation-sensitive gel electrophoresis. Furthermore, different DNA changes in the same PCR fragment could be distinguished by their peak patterns. CONCLUSIONS: Capillary-based HA is a fast, efficient, and sensitive method that considerably reduces the amount of "hands-on" time for each sample. By this approach, the entire coding regions of BRCA1 and BRCA2 from two breast cancer patients can be scanned in a single run of 90 min.

Autoanalysis↗

PubMed1996

Rapid detection of regionally clustered germ-line BRCA1 mutations by multiplex heteroduplex analysis. UKCCCR Familial Ovarian Cancer Study Group.

Germ-line mutations of the BRCA1 gene are responsible for a substantial proportion of families with multiple cases of early-onset breast and/or ovarian cancer. Since the isolation of BRCA1 last year, >65 distinct mutations scattered throughout the coding region have been detected, making analysis of the gene time consuming and technically challenging. We have developed a multiplex heteroduplex analysis that is designed to analyze one-quarter of the coding sequence in a single-step screening procedure and that will detect approximately 50% of all BRCA1 mutations so far reported in breast/ovarian cancer families. We have used this technique to analyze BRCA1 in 162 families with a history of breast and/or ovarian cancer and identified 12 distinct mutations in 35 families.

BRCA1 Protein↗

PubMed1984

The detection of subtle differences between different orthopoxvirus genomes by heteroduplex analysis.

Corresponding DNA fragments from variola (Harvey) and monkeypox (Denmark) viruses which had been cloned into different plasmid vectors were subjected to heteroduplex analysis. Characteristic deletion loops corresponding to differences between the cloning vectors served as internal markers to identify and to orientate the heteroduplexed molecules. Partial denaturation of the resulting heteroduplexes was used as a primary screen to locate regions of heterogeneity between the poxvirus inserts. The denaturation threshold for homoduplexes was consistently higher than that for heteroduplexes. However, significant sequence divergence between corresponding fragments was indicated by larger than usual differences in thresholds between corresponding homo- and heteroduplexes. Denaturation bubbles of 0.1-0.5 kb were detected and hence small regions of heterogeneity between the genomes (180 kb) of variola and monkeypox viruses were localised. This procedure has a general application in comparative studies on large, complex but closely related DNA molecules.

Base Sequence↗

PubMed1997

Hereditary coproporphyria: exon screening by heteroduplex analysis detects three novel mutations in the coproporphyrinogen oxidase gene.

Hereditary coproporphyria is a dominantly inherited disorder of porphyrin metabolism caused by a partial deficiency of coproporphyrinogen oxidase, the sixth enzyme in the heme synthetic pathway. We investigated the molecular basis of hereditary coproporphyria in three unrelated patients, amplifying each exon of the coproporphyrinogen oxidase gene and performing heteroduplex analysis to look for mutations. Unique heteroduplex patterns were noted in exons 2, 3, and 6. Sequencing revealed different mutations in each patient: a G-->A point mutation encoding a glutamic acid to lysine substitution at codon 101 (E101K), a C-->T point mutation encoding a proline to serine substitution at codon 149 (P149S), and a one base-pair insertion in exon 6 (968insT). No other mutations were found on sequencing the remaining exons and their intron-exon junctions. The two point mutations affect amino acids that are conserved in all species studied to date. The one base-pair insertion in exon 6 is the first frameshift mutation to be described in the coproporphyrinogen oxidase gene. This study adds three new mutations to those that have been previously reported, and all have been restricted to single families. These results indicate that hereditary coproporphyria is a genetically heterogeneous disease.

Amino Acid Sequence↗

PubMed1997

Characterization of insect cell lines: heteroduplex analysis employing a mitochondrial 16S ribosomal RNA gene fragment.

Routine cell line characterization procedures are not adequate for characterizing the cell lines of insect origin. Ribosomal RNA (rRNA) gene sequences and their comparisons have been used successfully for delineating species and phylogenetic analysis. Using similar principles, we have standardized a protocol for the confirmation of species identity of insect cell lines. The procedure includes PCR amplification of the mitochondrial 16S rRNA gene fragment from the cell line and larvae of known insect species and heteroduplex analysis to detect the sequence variation in the PCR-amplified rRNA gene fragments. If the PCR fragment of the cell lines yields a homoduplex with the larvae of known species, then the cell line is conspecific with the larvae. If the larvae and cell line are of two different species, then the analysis exhibits multiple bands of heteroduplexes. The technique also allows detection of cross-contamination of culture having two insect cell lines belonging to two different species.

Aedes↗