Analysis of single nucleotide polymorphisms in the promoter region of interleukin-10 by denaturing high-performance liquid chromatography.

Interleukin-10 (IL10), an anti-inflammatory cytokine, has been implicated in a variety of immune- and inflammatory-related diseases. We investigated the following SNPs: -1082, -819, -592 in the promoter region of IL10 in a normal (control) population and selected diseases: breast cancer (BrCa), systemic lupus erythematosus (SLE), and B-cell chronic lymphocytic leukemia (B-CLL) by denaturing high-performance liquid chromatography (DHPLC) and found distinct genotype and haplotype patterns. DHPLC was performed using the Transgenomic WAVE instrument, a mutational discovery tool that allows for high throughout analysis of SNPs. The principle of DHPLC is based on separation of homo- and heteroduplex formation of individual polymerase chain reaction products at specific melting temperatures and set gradients. The melting temperature selected for each SNP was based on size and sequence of the polymerase chain reaction product (for -1082, 57 degrees C; for -819, 58 degrees C; and for -592, 59.2 degrees C). Before fragment mutational analysis, all samples were denatured at 95 degrees C and slowly reannealed to allow for reassociation of different strands. Heteroduplex samples were easily distinguished from homoduplex samples. In order to identify wild type from homozygous mutant, two homoduplex polymerase chain reaction samples had to be mixed together, denatured at 95 degrees C and reannealed. The homozygous mutant, when combined with wild type, displayed a double peak on chromatogram. Once distinct chromatograms were established for each of the SNPs and the nucleotide changes confirmed by sequencing, genotype and haplotype frequencies were tabulated for the groups studied.     

Analysis of sequence alterations in a defined DNA region: comparison of temperature-modulated heteroduplex analysis and denaturing gradient gel electrophoresis

The ability to detect DNA sequence heterogeneity quickly and reliably is becoming increasingly important as more genes involved in disease processes are discovered. We have assessed the ability of a high pressure liquid chromatography technique (HPLC) termed temperature-modulated heteroduplex analysis (TMHA) to detect a collection of 20 point mutations distributed throughout a 279 base pair fragment spanning the exon 8 region of the human HPRT gene. All mutant/wild type heteroduplexes formed from mutations in the lowest temperature melting domain of the fragment were easily resolved from the corresponding mutant and wt homoduplexes, while those generated from mutants in the next higher melting domain barely resolved from their parental homoduplexes. For comparison, identical heteroduplex samples were subjected to denaturing gradient gel electrophoresis (DGGE). Heteroduplexes in the lowest temperature melting domain were easily resolved, while no resolution was achieved with those in the next higher melting domain. These results suggest that TMHA and DGGE are measuring similar melting characteristics in heteroduplex molecules. TMHA appears to be a robust approach for detecting and/or purifying a wide variety of mutations in a defined region of DNA, provided that the melting characteristics of the fragment under study are carefully considered.     

Prenatal and post-natal screening of β-thalassemia and hemoglobin E genes in Thailand using denaturing high performance liquid chromatography

We have developed methods based on PCR and denaturing high performance liquid chromatography (DHPLC) for rapid identifications of common β-thalassemia mutations found in Thailand. The β-globin gene was separately amplified by PCR on four different fragments covering eight most common β-thalassemia mutations including nucleotide ?28 A-G, codon 17 (A-T), IVSI-1 (G-T), IVSI-5 (G-C), codon 26 (G-A or Hb E), codons 41/42 (–TTCT), codons 71/72 (+A) and IVSII-654 (C-T). After PCR amplification, heteroduplex was generated by denaturation at 95 °C for 5 min followed by a slow reduction in temperature to 25 °C at 0.03 °C/s. Analysis of heteroduplex was done on an automated WAVE Nucleic Acid Fragment Analysis System. Specific DHPLC profile for each mutation was demonstrated which could be used in screening for all eight β-thalassemia mutations. Further validation was done on 42 pre- and post-natal DNA samples which demonstrated 100 % accuracy as compared to the result obtained with conventional PCR assays. In a remaining case with an unknown mutation, a different DHPLC profile was noted on one of the amplified fragment. Further DNA sequencing of this fragment revealed a T-G transversion at the IVSI-116, a previously un-described mutation in Thai population. The DHPLC assay developed should prove useful for rapid screening of known and unknown β-thalassemia mutations during carrier screening and pre-natal diagnosis which would facilitate an ongoing prevention and control program of thalassemia.     

Improvement of fragment and primer selection for mutation detection by denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) is one of the most powerful methods for mutation detection currently available. For successful application the appropriate selection of PCR fragments and PCR primers is crucial. The sequence of interest should always be within the domain with the lowest melting temperature. When more than one melting domain is present the fragment is generally divided into several smaller ones. This, however, is not always necessary. We found that simple modifications of PCR fragments and primer sequences may substantially reduce the number of amplicons required. Furthermore, by plotting the (natural) melting curves of fragments without a GC-clamp, we could explain why fragments theoretically perfect for DGGE in practice failed to reveal mutations. Alternative fragment selection and the use of modified primers (addition of T/A or G/C tails) result in the detection of mutations that originally remained undetected. Our studies extend the utility of DGGE by using a minimum of PCR fragments and achieving a maximum of mutation detection.     

Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis.

Duplex DNA fragments differing by single base substitutions can be separated by electrophoresis in denaturing gradient polyacrylamide gels, but only substitutions in a restricted part of the molecule lead to a separation (1). In an effort to circumvent this problem, we demonstrated that the melting properties and electrophoretic behavior of a 135 base pair DNA fragment containing a beta-globin promoter are changed by attaching a GC-rich sequence, called a 'GC-clamp' (2). We predicted that these changes should make it possible to resolve most, if not all, single base substitutions within fragments attached to the clamp. To test this possibility we examined the effect of several different single base substitutions on the electrophoretic behavior of the beta-globin promoter fragment in denaturing gradient gels. We find that the GC-clamp allows the separation of fragments containing substitutions throughout the promoter fragment. Many of these substitutions do not lead to a separation when the fragment is not attached to the clamp. Theoretical calculations and analysis of a large number of different mutations indicate that approximately 95% of all possible single base substitutions should be separable when attached to a GC-clamp.     

Denaturing high-performance liquid chromatography in the detection of ABCA1 gene mutations in familial HDL deficiency

Mutations in the ABCA1 gene are the cause of familial high density lipoprotein deficiency (FHD). Because these mutations are spread over the entire gene, their detection requires the sequencing of all 50 exons. The aim of this study was to validate denaturing high-performance liquid chromatography (DHPLC) in mutation detection as an alternative to systematic sequencing. Exons of the ABCA1 gene were amplified using primers employed for sequencing. Temperatures for DHPLC were deducted from a software and empirically defined for each amplicon. To assess DHPLC reliability, we tested 30 sequence variants found in FHD patients and controls. Combined DHPLC and sequencing was applied to the genotyping of new FHD patients. Most of the amplicons required from two to five temperature conditions to obtain partially denatured DNA over the entire amplicon length. Twenty-nine of the variants found by sequencing were detected by DHPLC (97% sensitivity). The detection of the last variant (in exon 40) required different primers and amplification conditions. DHPLC and sequencing analysis of new FHD patients revealed that all amplicons showing a heteroduplex DHPLC profile contained sequence variants. No variants were detected in amplicons with a homoduplex profile. DHPLC is a sensitive and reliable method for the detection of ABCA1 gene mutations.     

Denaturing high-performance liquid chromatography detects reliably BRCA1 and BRCA2 mutations

Denaturing high-performance liquid chromatography (DHPLC) is a recently developed method of comparative sequencing based upon heteroduplex detection. To assess the reliability of this method, 180 different mutations (54 deletions, 12 insertions, and 117 single base substitutions) in BRCA1 and BRCA2 were tested. Second, 25 index individuals with complete DHPLC analysis of BRCA1 were reanalyzed by dye-terminator sequencing. Third, 41 index individuals were analyzed concomitantly by both DGGE and DHPLC. Of the 180 different BRCA1 and BRCA2 mutations, 179 showed heterozygous DHPLC elution profiles. Dye-terminator sequencing of the entire BRCA1 gene, including 5592 bp of coding sequence and 5206 bp of flanking noncoding sequence, in 25 index individuals did not reveal additional variants missed by DHPLC. The concomitant analysis of 41 index cases showed that 4 probably disease-associated mutations were identified by DHPLC while only 3 of those 4 sites were detected by denaturing gradient gel electrophoresis. We conclude that DHPLC is a sensitive and cost-effective method for the screening of BRCA1 and BRCA2.     

Optimized PCR fragments for heteroduplex analysis of the whole human mitochondrial genome with denaturing HPLC

Denaturing high pressure liquid chromatography (dHPLC) is an efficient method for discovery of unknown mutations by heteroduplex analysis of PCR fragments. For comprehensive mutation scanning of the whole 16.569 bp human mitochondrial genome, we developed a set of 67 primer pairs defining overlapping PCR fragments that are well suited for heteroduplex analysis. The aim of our optimization efforts was to ensure that point mutations are detectable at every nucleotide position of each amplicon. Some GC-rich regions of mitochondrial DNA (mtDNA) were found to have unfavourable melting profiles in all possible amplicons, therefore requiring GC-clamps at the end of one or both oligonucleotide PCR primers. Following detection of a heteroduplex pattern by dHPLC, our primers can also be employed for DNA sequencing to identify the underlying mutation. In case of heteroplasmic mutations with a low proportion of mutant mtDNA, a fragment collector is useful to recover the heteroduplex peak, which contains mutant and wildtype DNA molecules in a 1:1 ratio.     

Temperature-mediated heteroduplex analysis for the detection of drug-resistant gene mutations in clinical isolates of Mycobacterium tuberculosis by denaturing HPLC, SURVEYOR nuclease

Denaturing high-performance liquid chromatography (DHPLC) is a relatively new technique, which utilizes heteroduplex formation between wild-type and mutated DNA strands to identify point mutations. Heteroduplex molecules are separated from homoduplex molecules by ion-pair, reverse-phase liquid chromatography on a special column matrix with partial heat denaturation of the DNA strands. In order to investigate the application of this method for point mutation detection in drug-resistant genes of Mycobacterium tuberculosis, katG, rpoB, embB, gyrA, pncA and rpsL genes, which are responsible for isoniazid, rifampicin, ethambutol, fluoroquinolone, pyrazinamide and streptomycin resistance, respectively, were detected by temperature-mediated DHPLC in 10 multidrug-resistant and 10 drug-susceptible clinical isolates. The DHPLC data were compared with those from a conventional MIC test. The results show that DHPLC is cost-effective with high capacity and accuracy, and is potentially useful for genotypic screening for mutations associated with anti-tuberculosis drug resistance.     

Rapid screening of the entire mitochondrial DNA for low-level heteroplasmic mutations

Alterations of the mitochondrial DNA (mtDNA) are implicated in various pathological conditions. In this study, we used denaturing high performance liquid chromatography (DHPLC) as a method to rapidly screen the entire mtDNA for mutations. Overlapping DNA fragments, amplified by one single cycling protocol from frozen pre-formulated PCR mixes, were subjected to DHPLC analysis. Single DHPLC injections of fragments yielded straightforward interpretation of results with a detection limit down to 1% mtDNA heteroplasmy. Furthermore, collection and re-amplification of low degree heteroduplex peak-fractions allowed sequence analysis of mtDNA mutations down to the detection limit of the DHPLC method. In order to demonstrate that the method has diagnostic value, we analyzed and confirmed known mtDNA mutations in patient samples.     

Absence of mutations in the promoter region of the low density lipoprotein receptor gene in a large number of familial hypercholesterolaemia patients as revealed by denaturing gradient gel electrophoresis

Summary Denaturing gradient gel electrophoresis (DGGE) was used in combination with the polymerase chain reaction (PCR) to detect sequence variations in the promoter region of the low density lipoprotein receptor (LDLR) gene. On the basis of calculated predictive melting properties we designed primers to amplify a 447-bp fragment of the promoter region from position-512 to -66, containing previously identified regulatory sequences. Using a primer with a GC-clamp in combination with restriction enzyme digestion, two melting domains could be analysed simultaneously. By oligonucleotide-directed mutagenesis artificial mutants were generated to optimize the conditions and to test the sensitivity of the method. All mutants were readily detected by electrophoresis in a 9% polyacrylamide gel containing a 10%–60% linear denaturing gradient. Using this method, we analysed DNA samples of 350 heterozygous familial hypercholesterolaemia (FH) patients. No mutations were detected, suggesting that mutations in the regulatory elements of the promoter sequence do not play a significant role in the etiology of FH.     

A program for selecting DNA fragments to detect mutations by denaturing gel electrophoresis methods.

A computer program was developed to automate the selection of DNA fragments for detecting mutations within a long DNA sequence by denaturing gel electrophoresis methods. The program, MELTSCAN, scans through a user specified DNA sequence calculating the melting behavior of overlapping DNA fragments covering the sequence. Melting characteristics of the fragments are analyzed to determine the best fragment for detecting mutations at each base pair position in the sequence. The calculation also determines the optimal fragment for detecting mutations within a user specified mutational hot spot region. The program is built around the statistical mechanical model of the DNA melting transition. The optimal fragment for a given position is selected using the criteria that its melting curve has at least two steps, the base pair position is in the fragment's lowest melting domain, and the melting domain has the smallest number of base pairs among fragments that meet the first two criteria. The program predicted fragments for detecting mutations in the cDNA and genomic DNA of the human p53 gene.     

DHPLC analysis of adenomatous polyposis coli (APC) mutations using ready-to-use APC plates: simple detection of multiple base pair deletion mutations

The adenomatous polyposis coli (APC), which is the susceptible gene for familial adenomatous polyposis (FAP) and sporadic colorectal cancer, spans 15 exons. The open reading frame of APC is 8529 bp, which encodes 2843 amino acids. Conventional genetic screening involves extensive time as well as high cost and labor. Thus, we developed a novel APC ready-to-use plate for high-throughput mutational analysis by denaturing high performance liquid chromatography (DHPLC). To prepare the ready-to-use APC plate, all 38 primer pairs and PCR mixtures were aliquoted into individual wells of a 96-well plate, and frozen at -20 degrees C until use. All 38 PCR primers were designed to be amplified at the same temperature (52 degrees C). We examined a total of 27 FAP patient samples with APC germline mutations (17 for multiple bp deletions, 1 for 1 bp deletion, 9 for nonsense mutations) and 50 APC-negative noncarriers. All 17 multiple bp deletion mutations were detected during the initial 50 degrees C running analysis and thus ruled out for further analyses. All other mutations were clearly detected under specific optimized conditions. More than 50% of the APC germline mutations were multiple base pair deletions and efficiently selected by omitting time-consuming partial denaturing conditions.     

Application and evaluation of denaturing HPLC for molecular genetic analysis in tuberous sclerosis

Tuberous sclerosis (TSC) is an autosomal dominant disorder characterised by the development of hamartomas in multiple tissues and organs. TSC exhibits locus heterogeneity with genes at 9q34 (TSC1) and 16p13.3 (TSC2) that have 21 and 41 coding exons, respectively. The mutational spectrum at both loci is wide and previous studies have shown that 60%-70% of cases are sporadic and represent new mutations. We have formatted denaturing high performance liquid chromatography (DHPLC) for rapid screening of all coding exons of TSC1 and TSC2. DHPLC analysis detected likely disease-causing mutations in 103 of 150 unrelated cases (68%), compared with 92/150 (61%) and 87/150 (58%) for single-strand conformation polymorphism analysis (SSCP) and conventional heteroduplex analysis (HA), respectively. Capital, consumable and labour costs were determined for each exon screening procedure. Estimated costs per patient sample depended on throughput, particularly for DHPLC, where a high proportion of costs are fixed, and were pounds sterling 257, pound sterling 216 and pound sterling 242 for DHPLC, SSCP and HA, respectively, assuming a throughput of 252 samples per year, or pound sterling 354, pound sterling 233 and pound sterling 259, assuming a throughput of 126 samples per year. DHPLC had the advantages of increased sensitivity and reduced labour costs when compared with more traditional approaches to exon screening but, unless expensive DHPLC equipment is being efficiently utilised for a very high proportion of the time available, overall costs are slightly higher.     

Denaturing high-performance liquid chromatography is a suitable method for PMM2 mutation screening in carbohydrate-deficient glycoprotein syndrome type IA patients

The phosphomannomutase 2 gene (PMM2; MIM 601785) has been identified as the carbohydrate-deficient glycoprotein syndrome type 1A gene (CDGS type 1A; MIM 212065). The gene spans 8 exons and 741 bp of coding DNA. Previously, we have identified 20 different mutations in the PMM2 gene using mutation screening with single-stranded conformation polymorphism (SSCP) and sequencing of DNA from 61 CDGS type 1A patients. Because eight of these could not be detected by SSCP, we were not satisfied with the sensitivity of the mutation detection technique used. Thus, we wanted to investigate if denaturing high-performance liquid chromatography (DHPLC) was a more suitable mutation screening method for PMM2. DHPLC was set up for PMM2 by optimizing eight different PCR fragments, one for each exon. The mutation detection was optimized empirically with PCR fragments from controls. First, control samples were run at a universal gradient and after modification and shortening of the gradient, also run at 10 different temperatures, 50-70 degrees C with 2-degree intervals, to enable setting of the temperature with the highest resolution. Then, PCR products with known mutations from the previous study were analyzed, and the results were compared to the control chromatograms for aberrations. We detected 19/20 mutations with DHPLC, and several mutations not detected by earlier screening techniques were readily detected by DHPLC. We conclude that DHPLC is a suitable detection technique for a rapid and reliable first scan of CDGS type 1A patients.     

Denaturing gradient gel method for mapping single base changes in human mitochondrial DNA.

A denaturing gradient gel electrophoresis (DGGE) method is described that detects even single base pair changes in mitochondrial DNA (mtDNA). In this method, restriction fragments of mtDNA are electrophoresed in a urea/formamide gradient gel at 60 degrees C. Migration distance of each mtDNA fragment in the gel depends on melting behavior which reflects base composition. Fragments are located by Southern blotting with specific mtDNA probes. With just four carefully chosen restriction enzymes and as little as 50-100 ng of mtDNA, the method covers almost the entire human mitochondrial genome. To demonstrate the method, human mtDNA was analyzed. In six normal individuals, DGGE revealed melting behavior polymorphisms (MBPs) in mtDNA fragments that were not detected by restriction fragment length polymorphism (RFLP) analysis in agarose gels. Another individual, shown to have a melting behavior polymorphism in the cytochrome b coding region, was studied in detail. By mapping, the mutation was deduced to lie between nt 14905 and 15370. The affected fragment was amplified by PCR and sequenced. Specific base changes were identified in the region predicted by the gel result. This method will be especially useful as a diagnostic tool in mitochondrial disease for rapid localization of mtDNA mutations to specific regions of the genome, but DGGE also could complement RFLP analysis as a more sensitive method to follow maternal lineage in human and animal populations in a variety of research fields.     

Denaturing High-Performance Liquid Chromatography Detects Reliably BRCA1 and BRCA2 Mutations

Denaturing high-performance liquid chromatography (DHPLC) is a recently developed method of comparative sequencing based upon heteroduplex detection. To assess the reliability of this method, 180 different mutations (54 deletions, 12 insertions, and 117 single base substitutions) in BRCA1 and BRCA2 were tested. Second, 25 index individuals with complete DHPLC analysis of BRCA1 were reanalyzed by dye-terminator sequencing. Third, 41 index individuals were analyzed concomitantly by both DGGE and DHPLC. Of the 180 different BRCA1 and BRCA2 mutations, 179 showed heterozygous DHPLC elution profiles. Dye-terminator sequencing of the entire BRCA1 gene, including 5592 bp of coding sequence and 5206 bp of flanking noncoding sequence, in 25 index individuals did not reveal additional variants missed by DHPLC. The concomitant analysis of 41 index cases showed that 4 probably disease-associated mutations were identified by DHPLC while only 3 of those 4 sites were detected by denaturing gradient gel electrophoresis. We conclude that DHPLC is a sensitive and cost-effective method for the screening of BRCA1 and BRCA2.     

DHPLC mutation analysis of the hereditary nonpolyposis colon cancer (HNPCC) genes hMLH1 and hMSH2

Denaturing high-performance liquid chromatography (DHPLC) is an efficient method for detection of mutations involving a single or few numbers of nucleotides, and it has been successfully used for mutation detection in disease-related genes. Colorectal cancer is one of the most common cancers, and mutations in the genes for hereditary nonpolyposis colon cancer (HNPCC), hMLH1 and hMSH2, also involve mainly point mutations. Sequence analysis is supposed to be a screening method with high sensitivity; however, it is time-consuming and expensive. We therefore decided to test sensitivity and reproducibility of DHPLC for 71 sequence variants in hMLH1 and hMSH2 initially found by sequence analysis in DNA samples of German HNPCC patients. DHPLC conditions of the PCR products were based on the melting pattern of the wild-type sequence of the corresponding PCR fragments. All but one of the 71 mutations was detected using DHPLC (sensitivity of 97%). Running time per sample averaged only 7 min, and the system is highly automated. Thus DHPLC is a rapid and sensitive method for the detection of hMLH1 and hMSH2 sequence variants.     

Unexpected behavior of H2Kb mutant DNAs in denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) is based upon the different melting behaviors of DNA molecules in a chemical denaturant gradient according to their sequences. This technique has recently become a widespread tool to detect mutations. The introduction of a GC-clamp enables the detection of most single base differences between two DNA molecules. As a test system we have applied the polymerase chain reaction (PCR) in combination with DGGE to detect a number of mutations in the mouse H2Kb DNA sequence. A wide variety of spontaneous in vivo mutations of this haplotype have been reported in the C57BL/6J mouse strain and are clustered in the alpha 1 and alpha 2 domains. The combination of PCR and DGGE revealed almost all base changes present in the H2Kb mutants used. However, most of the PCR products of these mutants showed melting behavior which is not easily predicted. We suggest that in addition to current simple theory, which considers that the migration of a DNA molecule in a denaturing gradient depends primarily on its initial melting behavior, additional factors such as secondary structure in partially melted molecules may play a role and can be used to detect mutations.     

Modification of the melting properties of duplex DNA by attachment of a GC-rich DNA sequence as determined by denaturing gradient gel electrophoresis.

The melting behavior of a DNA fragment carrying the mouse beta maj-globin promoter was investigated as a means of establishing procedures for separating DNA fragments differing by any single base substitution using the denaturing gradient gel electrophoresis procedure of Fischer and Lerman (1,2). We find that attachment of a 300 base pair GC-rich DNA sequence, termed a GC-clamp, to a 135 bp DNA fragment carrying the mouse beta-globin promoter significantly alters the pattern of DNA melting within the promoter. When the promoter is attached to the clamp, the promoter sequences melt without undergoing strand dissociation. The calculated distribution of melting domains within the promoter differs markedly according to the relative orientation of the clamp and promoter sequences. We find that the behavior of DNA fragments containing the promoter and clamp sequences on denaturing gradient polyacrylamide gels is in close agreement with the theoretical melting calculations. These studies provide the basis for critical evaluation of the parameters for DNA melting calculations, and they establish conditions for determining whether all single base substitutions within the promoter can be separated on denaturing gradient gels.