Genotyping single nucleotide polymorphisms by primer extension and high performance liquid chromatography

We have investigated the possibility of genotyping single nucleotide polymorphisms (SNPs) by primer extension and high performance liquid chromatography (HPLC). Using three polymorphisms of current interest to our group (an A/G polymorphism in the proneurotensin gene and A/G and T/C polymorphisms in the 5HT2a receptor gene), we show that robust signal is obtained using this simple analytic method which has the added advantages that sample loading and analysis are essentially automated, analytic time is brief, and no further purification step after primer extension is required. We also show that all stages of the HPLC-primer extension genotyping can be multiplexed which, together with automation, suggests that this system may be suitable for linkage studies based upon emerging SNP maps. Received: 27 April 1998 / Accepted: 26 November 1998     

Simultaneous detection of CpG methylation and single nucleotide polymorphism by denaturing high performance liquid chromatography

We report here a novel method to simultaneously detect CpG methylation and single nucleotide polymorphisms (SNPs) using denaturing high performance liquid chromatography (DHPLC). PCR products of bisulfite-modified CpG islands were separated using DHPLC. BstUI digestion and DNA sequencing were used in confirmation studies. Consistent with the BstUI digestion assay, the 294 bp PCR product of the modified hMLH1 promoter showed different retention times between the methylated cell lines (RKO and Cla, 6.7 min) and the unmethylated cell lines (PACM82 and MGC803, 6.2 min). No hMLH1 methylation was observed in 13 primary gastric carcinomas and their matched normal tissues. One hMLH1 SNP was detected in gastric cancer patients, in both cancer and normal tissues. DNA sequencing revealed that the SNP is a G→A variation at –93 nt of the hMLH1 promoter. A two-peak chromatogram was also obtained in the 605 bp PCR product of the Cox-2 promoter of the AGS, HEK293 and MKN45 cell lines by DHPLC. Another peak corresponding to methylated CpG islands was observed on the chromatogram of the Cox-2-methylated AGS cell line after bisulfite treatment. In conclusion, methylation in homoallelic and heteroallelic CpG islands could be detected rapidly and reliably by bisulfite–DHPLC. A SNP in the target sequence could also be detected at the same time.     

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.     

Application of denaturing high-performance liquid chromatography for mapping of single nucleotide polymorphisms in barley (Hordeum vulgare L.).

Recent advances in DNA sequence analysis and the establishment of high-throughput assays have provided the framework for large-scale discovery and analysis of DNA sequence variation. In this context, single nucleotide polymorphisms (SNPs) are of particular interest. To initiate a systematic approach to develop an SNP map of barley (Hordeum vulgare L.), we have employed denaturing high-performance liquid chromatography (DHPLC) to analyse segregating SNP patterns in a doubled-haploid (DH) mapping population. To this end, SNPs between the parental genotypes were identified using a direct sequencing approach. Once a SNP was established between the parents, the optimal melting temperature of the PCR fragment containing the SNP was predicted for its analysis by DHPLC. Following the detection of the optimal temperature, the DH lines were analysed for the presence of either of the alleles. To test the utility of the analysis, data from previously mapped RFLP markers from which these SNPs were derived were compared. Results from these experiments indicate that DHPLC can be efficiently employed in analysing SNPs on a high-throughput scale.     

Genomic organization and polymorphisms detected by denaturing high-performance liquid chromatography of porcine SLC11A1 gene.

SLC11A1 (also known as Natural Resistance Associated Macrophage Protein1, NRAMP1) plays a crucial role in resistance of inbred mice to infection with several intracellular pathogens such as Mycobacterium, Leishmania and Salmonella. In this study, PCR amplification and sequencing were performed to obtain the genomic organization and sequence of porcine SLC11A1 gene by comparative genomic analysis. Results showed that porcine SLC11A1 gene consists of 15 exons and 14 introns, which is consistent with that of mice and human. All introns were sequenced and their nucleotide sequences were submitted to GenBank. The exon/intron boundaries were determined by comparing cDNA sequence with amplified genomic DNA sequences. Mutational analysis was performed on exonic and neighboring intronic region by denaturing high-performance liquid chromatography (DHPLC) and sequencing confirmation. Forty polymorphisms were identified; six are located in exons and thirty-four in introns. Two exonic polymorphisms are nonsynonymous changes (D6H and V175I), three are synonymous changes (S23, G33 and I155), and one is in 3' UTR. The availability of the fine genomic organization and identification of the polymorphisms will facilitate the evaluation of porcine SLC11A1 functional role in diseases resistance or susceptibility.     

Molecular monitoring of the intestinal flora by denaturing high performance liquid chromatography

Gut flora analysis is hampered by the complexity of the intestinal microbiota and by inherent limitations of culture-based approaches. Therefore, culture-independent molecular methods based upon 16S rRNA gene analysis were applied successfully for the analysis of complex microbial communities. However, generally accepted and validated profiling methods such as denaturing and temperature gradient gel electrophoresis (DGGE/TGGE) are still laborious and time consuming. Thus, we adapted the separation of amplified bacterial 16S rRNA gene fragments by denaturing high performance liquid chromatography (DHPLC) using the WAVE Microbial Analysis System as a rapid and convenient means to display complex intestinal bacterial communities and to monitor changes in the gut flora. The separation of 16S rRNA gene fragments amplified from reference strains representing main gut bacterial populations and from human stool samples revealed that DHPLC analysis effectively detects bacterial groups predominant in the human gut flora. The investigation of faecal samples from hospitalized patients before, during and after antibiotic therapy showed that PCR-based DHPLC can be used to monitor gut flora changes. Results from DHPLC analysis were comparable with DGGE profiles generated from the same samples, demonstrating that the adapted DHPLC protocol is well suited for the analysis of complex microbial communities.     

Identification by denaturing high-performance liquid chromatography of numerous polymorphisms in a candidate region for multiple sclerosis susceptibility

Genetic association analysis of candidate regions where evidence of linkage has accumulated is becoming a key issue in the study of complex diseases. A high density of markers, at least one per centimorgan, is required to improve the chances of observing linkage disequilibrium with disease alleles. A recently available single nucleotide polymorphism (SNP) map designed to cover the whole genome provides an average density of one marker per 2 cM. In the present study we show that the number of markers can be approximately doubled in a selected region, thus reaching a density suitable for association studies, by applying a completely automated technique for polymorphism detection, denaturing high-performance liquid chromatography (DHPLC). A systematic search for SNPs was performed in the region 5ptel-q13, where weak but convergent evidence for linkage with multiple sclerosis has accumulated. Screening for polymorphisms was performed on 124 sequence tagged sites (STSs) in the 3'UTR ends of expressed sequence tags totaling about 30,000 bp. Thirty SNPs in 28 STSs were found with less than 10% overlap with the markers already detected in the same region. The data confirm the validity of the approach using DHPLC on expressed gene sequences tagged by a set of standard commercially available primers.     

Identification of norepinephrine in bovine oviductal fluid by high performance liquid chromatography.

The final stages of sperm maturation, fertilization and early embryonic development take place in the microenvironment of the oviduct and are essential for successful reproduction in mammals. Although catecholamines have been shown to have beneficial effects on mammalian gametes in vitro, identification of catecholamines in native bovine oviductal fluid has not been studied. The objective of this research was to identify catecholamines in bovine oviductal fluid and to determine whether concentrations of catecholamines change with stage of the estrous cycle. Oviductal fluid was collected via indwelling oviductal cannulae and assayed for the presence of catecholamines by high performance liquid chromatography. Norepinephrine was the only catecholamine detected, in concentrations ranging from 0.828 ng/ml - 1117 ng/ml. The presence of norepinephrine in oviductal fluid corresponded to a period of time just prior to, during, and after ovulation, when serum progesterone levels were low. This was a consistent finding in ODF collected from normally cycling cows. Potential functions of norepinephrine in oviductal fluid include regulation of fluid formation, induction of capacitation and the acrosome reaction in spermatozoa, and cleavage of the early embryo.     

Detection of differential gene copy number using denaturing high performance liquid chromatography

Genomic rearrangements leading to deletion or duplication of gene(s) resulting in alterations in gene copy number underlie the molecular lesion in several genetic disorders. Methods currently used to determine gene copy number including real time PCR, southern hybridization, fluorescence in situ hybridization, densitometric scanning of PCR product etc. have certain disadvantages and are also expensive and time consuming. Herein, we describe a simple and rapid method to assess gene copy number using denaturing high performance liquid chromatography (dHPLC). We used X chromosome genes as model to compare the gene copy numbers present on this chromosome in males and females. DNA from these samples were amplified by biplex PCR using primer pairs specific for X chromosome genes only (target gene) and for genes present on both X and Y chromosomes (internal control). Amplified products were analyzed using HPLC under non-denaturing conditions. The ratio of peak areas (target gene/internal control) of the amplified products was approximately twice in female samples than male samples (p < 0.001) demonstrating that the differential gene copy number can be easily detected using this method. This method can potentially be used for diagnostic purpose where the need is to distinguish samples based on the differential gene copy numbers.     

Detection of novel ALAD gene polymorphisms using denaturing high-performance liquid chromatography.

Denaturing high-performance liquid chromatography (DH-PLC), which is based on the separation of mismatched DNA heteroduplexes, is one of the most promising techniques for detecting nucleotide polymorphisms. Lead is an important environmental toxicant that can impair the cardiovascular, central nervous, renal, reproductive, and hematologic systems. Here we compare the sensitivity and efficiency of DNA polymorphism detection in the delta-aminolevulinate dehydratase (ALAD) gene encoding the principal lead-binding protein in humans by means of DHPLC and direct DNA sequencing of polymerase chain reaction amplicons. In a sample of 48 unrelated Chinese women, five novel mutations were discovered in intron 6 (G13298C). exon 7 (C13348T), intron 8 (C13847T), intron 12 (C15096T), and the 3' untranslated region of exon 13 (A15762C). The allele frequencies of C13298, T13348, T13847, T15096, and C15762 alleles were 21.3%, 2.3%. 82.1%, 62.5%, and 1.1%, respectively. All five mutations were detected by both DHPLC and direct DNA sequencing. No previously reported missense ALAD mutations were found in this Chinese population. Our study confirms that DHPLC provides an accurate method for the rapid identification of single nucleotide polymorphisms.     

Comprehensive screening of the thiopurine methyltransferase polymorphisms by denaturing high-performance liquid chromatography

The drug-metabolizing enzyme thiopurine S-methyltransferase (TPMT) catalyzes the S-methylation of thiopurines such as 6-mercaptopurine, 6-thioguanine, and azathiopurine, which are used as immunosuppressants and in the treatment of acute lymphoblastic leukemia and rheumatoid arthritis. TPMT enzymatic activity is a polymorphic trait, and poor metabolizers may develop life-threatening bone marrow failure. To avoid such adverse effects, the TPMT enzymatic activity in patients' red blood cells (RBCs) is routinely measured prior to thiopurine administration in a limited number of oncology clinics. In the present study, we took advantage of a highly sensitive and specific automated denaturing high-performance liquid chromatography (dHPLC) technique that not only detects known polymorphic alleles, but also identifies previously uncharacterized sequence variants. We developed a dHPLC-based protocol to analyze the entire coding region and validated the protocol to detect all 16 previously described variant alleles. We further analyzed the entire coding region of the TPMT gene in 288 control samples collected worldwide and identified two novel amino acid substitutions Arg163Cys (487C>T) and Arg226Gln (677G>A) within exons 7 and 10, respectively. The clinical application of this comprehensive screening system for examining the entire TPMT gene would help to identify patients at risk for bone marrow failure prior to 6-mercaptopurine therapy.     

Mutation analysis of the entire mitochondrial genome using denaturing high performance liquid chromatography

In patients with mitochondrial disease a continuously increasing number of mitochondrial DNA (mtDNA) mutations and polymorphisms have been identified. Most pathogenic mtDNA mutations are heteroplasmic, resulting in heteroduplexes after PCR amplification of mtDNA. To detect these heteroduplexes, we used the technique of denaturing high performance liquid chromatography (DHPLC). The complete mitochondrial genome was amplified in 13 fragments of 1–2 kb, digested in fragments of 90–600 bp and resolved at their optimal melting temperature. The sensitivity of the DHPLC system was high with a lowest detection of 0.5% for the A8344G mutation. The muscle mtDNA from six patients with mitochondrial disease was screened and three mutations were identified. The first patient with a limb-girdle-type myopathy carried an A3302G substitution in the tRNA^Leu(UUR) gene (70% heteroplasmy), the second patient with mitochondrial myopathy and cardiomyopathy carried a T3271C mutation in the tRNA^Leu(UUR) gene (80% heteroplasmy) and the third patient with Leigh syndrome carried a T9176C mutation in the ATPase6 gene (93% heteroplasmy). We conclude that DHPLC analysis is a sensitive and specific method to detect heteroplasmic mtDNA mutations. The entire automatic procedure can be completed within 2 days and can also be applied to exclude mtDNA involvement, providing a basis for subsequent investigation of nuclear genes.     

High-throughput SNP detection by using DNA pooling and denaturing high performance liquid chromatography (DHPLC)

One of the critical steps in the positional cloning of a complex disease gene involves association analysis between a phenotype and a set of densely spaced diallelic markers, typically single nucleotide repeats (SNPs), covering the region of interest. However, the effort and cost of detecting sufficient numbers of SNPs across relatively large physical distances represents a significant rate-limiting step. We have explored DNA pooling, in conjunction with denaturing high performance liquid chromatography (DHPLC), as a possible strategy for augmenting the efficiency, economy, and throughput of SNP detection. DHPLC is traditionally used to detect variants in polymerase chain reaction products containing both allelic forms of a polymorphism (e.g., heterozygotes or a 1:1 mix of both alleles) via heteroduplex separation and thereby requires separate analyses of multiple individual test samples. We have adapted this technology to identify variants in pooled DNA. To evaluate the utility and sensitivity of this approach, we constructed DNA pools comprised of 20 previously genotyped individuals with a frequency representation of 0%-50% for the variant allele. Mutation detection was performed by using temperature-modulated heteroduplex formation/DHPLC and dye-terminator sequencing. Using DHPLC, we could consistently detect SNPs at lower than 5% frequency, corresponding to the detection of one variant allele in a pool of 20 alleles. In contrast, fluorescent sequencing detected variants in the same pools only if the frequency of the less common allele was at least 10%. We conclude that DNA pooling of samples for DHPLC analysis is an effective way to increase throughput efficiency of SNP detection.     

Assay of bovine fibrinopeptides by high performance liquid chromatography.

A method for separating small amounts (<10^?5 mol) of bovine fibrinopeptides A and B employing high performance liquid chromatography has been developed. The limit of detectability of this method is about 10^?10 mol of fibrinopeptide. The separation was achieved within 20 min under reversed phase conditions using isocratic elution with aqueous buffer-acetonitrile solvent systems.     

Molecular size characterization of bacterial capsular polysaccharide vaccines by high performance liquid chromatography

Bacterial capsular polysaccharides are major virulence factors and some are used as vaccinal antigens. Their molecular size is an important physicochemical criterion which correlates with immunogenicity. This article describes a new application of high performance liquid chromatography (HPLC), based on molecular sieving, for such an evaluation. This HPLC method is rapid, accurate, reproducible, requires only very low amounts of product and presents good correlation with conventional gel permeation chromatography.     

Genetic variability of immunomodulatory genes in ectromelia virus isolates detected by denaturing high-performance liquid chromatography

The genetic variability of nine genes in 12 isolates and strains of ectromelia virus, which causes a smallpox-like disease (mousepox) in mice, was determined and allows for classification of ectromelia viruses. The low genetic variability suggests that evolutionary pressure maintains the activity of immunomodulatory genes in natural poxvirus infections.