Denaturing High-performance Liquid Chromatography Method Research Articles

Optimizing denaturing HPLC as a robust technique for identification of Short Tandem Repeats (STR) in forensic medicine

IntroductionShort Tandem Repeats (STRs) are defined as short lengths of 2–7 base pairs spreading through human genome which due to their highly diverse individually distribution are widely applied for identity detection and other forensic medicine purposes. Burdening considerable costs by the conventional methods such as capillary electrophoresis, we aimed to compare concomitant usage of multiplex PCR and denaturing high-performance liquid chromatography (DHPLC) as cheap, fast, highly accurate, and more accessible methods, with capillary electrophoresis (CE) to evaluate their potential for early screening of STRs. Materials and methodsThe present study randomly included 20 blood samples from the subjects referred to forensic medicine of Semnan, Iran. According to the size and allele frequency, we selected 8 major STR loci including CSF1PO, VWA, D18S51, TPOX, Amelogenin, FGA, SE33, and Penta D. A quad-STR multiplex PCR was performed for each locus and the PCR products were then analyzed using DHPLC machine and compared with the basic genetic properties obtained by capillary electrophoresis. ResultsBy optimizing the PCR and DHPLC conditions, our findings suggest this strategy as an effective method for STR detection. The genotypes were determined using size of loci which led to comparable results with capillary electrophoresis confirming an insignificant variation in the detection of TOPX, Amelogenin, CSF1PO, and D18S5 (p = 0.331), but discrepant results for FGA and VWA loci (p = 0.002). ConclusionOur study proposed DHPLC method as an effective screening method to characterize TOPX, Amelogenin, CSF1PO, and D18S51 as frequently used STR loci during identity detection in forensic medicine.

Setup of a Protocol of Molecular Diagnosis of β-Thalassemia Mutations in Tunisia using Denaturing High-Performance Liquid Chromatography (DHPLC).

β-Thalassemia is one of the most prevalent worldwide autosomal recessive disorders. It presents a great molecular heterogeneity resulting from more than 200 causative mutations in the β-globin gene. In Tunisia, β-thalassemia represents the most prevalent monogenic hemoglobin disorder with 2.21% of carriers. Efficient and reliable mutation-screening methods are essential in order to establish appropriate prevention programs for at risk couples. The aim of the present study is to develop an efficient method based on the denaturing high-performance liquid chromatography (DHPLC) in which the whole β-globin gene (HBB) is screened for mutations covering about 90% of the spectrum. We have performed the validation of a DHPLC assay for direct genotyping of 11 known β-thalassemia mutations in the Tunisian population. DHPLC assay was established based on the analysis of 62 archival β-thalassemia samples previously genotyped then validated with full concordance on 50 tests with blind randomized samples previously genotyped with DNA sequencing and with 96% of consistency on 40 samples as a prospective study. Compared to other genotyping techniques, the DHPLC method can meet the requirements of direct genotyping of known β-thalassemia mutations in Tunisia and to be applied as a powerful tool for the genetic screening of prenatal and postnatal individuals.

Identification of exon 19 and 21 mutations of EGFR gene in Chinese patients with esophageal squamous cell carcinoma

BackgroundAlthough epidermal growth factor receptor (EGFR) inhibitor treatment showed modest response in several clinical trials in esophageal squamous cell carcinoma (ESCC) patients, it has been reported that the frequency of EGFR mutations varied largely. The aim of this study was to investigate the existence of EGFR mutations in Chinese esophageal squamous cell carcinomas.MethodsFormalin-fixed paraffin-embedded surgically resected tumor samples were obtained from 127 randomly selected Chinese patients with ESCC. The most common EGFR mutations, including in-frame deletions in exon 19 and base substitutions in exon 21, were detected by denaturing high performance liquid chromatography (DHPLC) and direct sequencing simultaneously. K-RAS mutations in codons 12 and 13 were detected by direct sequencing.ResultsIn this study, L858R missense mutations of the EGFR gene were found in 8 out of 127 patients (6.3%) by DHPLC but no mutation was observed by direct sequencing. In addition, K-RAS mutation was detected in 2 out of 127 (1.6%) patients by direct sequencing.ConclusionsThe incidence of EGFR mutations was relatively high using DHPLC method but no mutation with direct sequencing in Chinese ESCC patients.

Establishment of a medium‐throughput approach for the genotyping of RHD variants and report of nine novel rare alleles

The routinely used serologic methods are robust in accurately typing standard D- or D+ blood. However, they result in discrepancy in weak or partial D blood, which requires genetic analysis. We have previously used denaturing high-performance liquid chromatography (DHPLC) to screen the entire RHD-coding sequence. However, DHPLC is technically challenging, labor-intensive, and time-consuming. To overcome these inconveniences, we sought to develop a new two-step approach. A total of 430 blood samples with D phenotype ambiguity were recruited for this study. The three most frequent weak D alleles (i.e., weak D, Type 1; weak D, Type 2; and weak D, Type 3), which altogether account for 60% to 90% of the atypical RHD alleles in the Caucasian population, were first identified by Tm-shift genotyping. The remaining unidentified samples were then subjected to a single-tube multiplex polymerase chain reaction (PCR) amplification of all 10 RHD exons followed by direct sequencing. Optimal conditions for efficient and reliable identification of the three most common weak D variants by Tm-shift genotyping were established. All 10 RHD exons were successfully amplified in a single-multiplex PCR procedure. Employment of the two-step analysis identified RHD variants in 91.6% of the 430 studied samples. Two of the nine previously undescribed variants, c.335G>T and c.939G>A, were found to cause aberrant mRNA splicing by means of a splicing minigene assay. The new two-step analysis proved to be much easier and cheaper than the DHPLC method and therefore is convenient to be used as a routine, medium-throughput approach for RHD genotyping.

Population-based case–control study on DAPK1, RAR-β2 and MGMT methylation in liquid-based cytology

Methylated genes have the potential to provide a new generation of cancer biomarkers. The aim of this study was to investigate: (1) the promoter methylation status of DAPK1, RAR-β2 and MGMT in randomly selected normal cytology of the general female population; (2) the effectiveness of gene methylation in liquid-based cytology to help in the early diagnosis of HSIL; (3) the relationship between HPV infection and gene methylation. Methylation of DAPK1, RAR-β2 and MGMT in 667 cervical samples with 331 cases of abnormal cytology and 336 randomly selected normal cytology controls was detected by methylation-specific PCR and denaturing high-performance liquid chromatography method (MSP-DHPLC). The methylation frequencies of each gene were compared. Methylation frequencies for MGMT, RAR-β2 and DAPK1 in normal cytology were 36.9, 42.0 and 46.7%, respectively. There was a trend toward increasing methylation frequency for any gene with age (p = 0.0133). Among abnormal cytology, there was a trend toward increasing number of methylation of any gene with severity of cytology grade (r = 0.1178, p = 0.0026). Methylation frequencies for MGMT and RAR-β2 among cytology of each grade were significantly different (χ ( 2 ) = 6.8976, p = 0.0086; χ ( 2 ) = 33.2477, p < 0.0001), and methylation frequencies for RAR-β2 in ASC were significantly higher than that in negative cytology (χ ( 2 ) = 8.7128, p = 0.0032). The relationship between MGMT, RAR-β2 and DAPK1 gene methylation and HPV infection was not found. This study reported methylation frequencies for MGMT, RAR-β2 and DAPK1 in normal cytology of the general female population. The combination of MGMT methylation, cytology and HPV infection is preferable for early detection of CIN2+ in cytology samples. There was no relationship between MGMT, RAR-β2 and DAPK1 gene methylation and HPV infection.

등온증폭반응법과 변성 고성능 액체 크로마토그래피를 이용한 B형 간염 바이러스의 검출

형광 검출기를 기반으로 한 변성 고성능 액체 크로마토그래피(DHPLC)분석법은 핵산검출에 유용하게 사용되고 있으며 또한 등온증폭 반응법은 병원성 미생물의 효과적인 검출방법으로 알려져 있다. 이 연구에서는 HBV의 조기 분석 방법으로써 사용되는 등온증폭반응법(LAMP)과 변성고성능 액체 크로마토그래피(DHPLC)의 검출한계와 특이성, 그리고 민감도를 평가하였다. 등온증폭 반응법의 검출 시간이 가장 빠른 장점을 보였으나, 변성 고성능 액체 크로마토그래피 분석법이 등온증폭반응법과 real-time PCR분석법과 비교한 결과, 10배 이상의 높은 민감도를 확인 할 수 있었다. 본 결과로서 B형 간염 바이러스의 진단을 위하여, 빠른 검출법으로써 등온증폭 반응법이 유용하게 쓰일 수 있을 것이며 변성 고성능 액체 크로마토그래피 분석법은 좀 더 낮은 병원균의 감염도 검출할 수 있어, 임상에서 유용하게 사용할 수 있을 것이다. The denaturing high performance liquid chromatography(DHPLC) with fluorescence detector assay is very useful tool for detecting nucleic acids. Furthermore, loop-mediated isothermal amplification(LAMP) constitutes a potentially valuable tool for rapid diagnosis of pathogenic microorganisms. In this study, we evaluated the specificity, detection limit, and sensitivity of a LAMP method and DHPLC method for rapid detection of the hepatitis b virus(HBV). As a result, the LAMP assay reported here has the advantage of rapid detection whereas, DHPLC assay has more sensitivity than other assays. These findings suggest that LAMP and DHPLC assay may be good tool for rapid diagnosis of clinical HBV infection.

Comparison of different methods for detecting epidermal growth factor receptor mutations in peripheral blood as a predictor of response to gefitinib.

10557 Background: Several studies have reported the detection of active EGFR mutation DNA in peripheral blood can also predict the response to EGFR-TKI in NSCLC. But heterogeneity in methodologies makes it difficult to use in daily practice. Goal of this study is to compare different methods for detecting EGFR mutation DNA in peripheral blood. Methods: From July 2007 to April 2009, fifty-one advanced NSCLC patients with pairs of tumour and serum samples treated with gefitinib were included. Response was evaluated by RECIST criteria. EGFR mutations in tumor tissues were detected by Scorpions-Amplification Refractory Mutation System (Scorpions-ARMS). Three methods were used to detect the mutations DNA in peripheral blood: denaturing high performance liquid chromatography (DHPLC), Mutant-Enriched-Liquidchip (ME-Liquidchip) and Scorpions-ARMS. Results: The Objective response rates (ORR) in whole group patients are 33.3% (17/51). EGFR Mutation rate were identified 15.7% (8/51), 27.5% (14/51), 29.4% (15/51) by DHPLC, ME-Liquidchip, Scorpions-ARMS in peripheral blood respectively. Only 34 cases were available for paired tumor sample EGFR mutation detection. In these 34 patients, EGFR mutation rate in tumor tissue was 41.2% (14/34). The corresponding rates of EGFR mutation status in peripheral blood with tumor tissues are 58.8% (by DHPLC), 58.8% (by ME-Liquidchip) and 70.6% (by Scorpions-ARMS). The ORRs by different method of EGFR mutation detection were 71.4% (Tumor sample), 62.5% (Blood DHPLC), 50.0% (Blood, ME-Liquidchip) and 66.7% (Blood, Scorpions-ARMS). EGFR Mutation detected by Scorpions-ARMS in tumor tissues and peripheral blood have a better prediction of ORR of EGFR-TKI (p=0.002 and p=0.003) compare DHPLC and ME-Liquidchip method. Conclusions: Tumor tissue is the better source of samples to detect EGFR mutation of NSCLC patients than peripheral blood. Scorpions-ARMS method is superior to DHPLC and ME-Liquidchip in serum EGFR mutation DNA detection. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Innovation Centre China, R&D AstraZeneca, Surexam, Inc AstraZeneca, GlaxoSmithKline AstraZeneca, Bayer, Novartis, sanofi-aventis

변성 고성능 액체 크로마토그래피를 이용한 한우, 젖소 그리고 혼입육의 구분

ABSTRACT. In the meat industry, correct breed information in food labeling is required to assure meat quality. Genetic markers provide corroborating evidence to identify breed. We described the development of DNA markers to discriminate between Korean beef cattle (Hanwoo), Holstein, and mixed cow beefs. As most breeds are standardized for coat colour, the melanocortin 1 receptor (MC1R) gene, involved in the regulation of eu/pheomelanins synthesis, has been suggested as marker for breed traceability of products of animal origin. We also designed sex-determining region Y (SRY) gene specific primers for Y chromosome detection. In thisstudy, fragments of MC1R gene and SRY gene were amplified by multiplex-PCR and subjected to digestion by Msp A1I restriction endonuclease. Reaction products were analysised by denaturing high performance liquid chromatography (DHPLC). As a result, we identified 6 DHPLC peak types from MC1R gene and SRY gene analysis. DHPLC method showed more sensitive than RFLP method for DNA fragments analysis. Therefore, DHPLC method can apply to identify for Hanwoo, Holstein and mixed beef.

Application of the DHPLC method for mutational detection of the CYP21A2 gene in congenital adrenal hyperplasia

Background More than 90% of cases of congenital adrenal hyperplasia (CAH) are caused by a steroid 21-hydroxylase deficiency. Approximately 75% of the defective CYP21A2 genes are generated through an intergenic recombination with the neighboring CYP21A1P pseudogene. These 2 duplicated genes share a 98% nucleotide sequence homology. Therefore, precisely identifying the CYP21A2 gene in CAH patients is absolutely necessary. Methods We describe an established PCR-based amplification method, a denaturing high-performance liquid chromatography (DHPLC) analysis, to directly identify 11 different mutations commonly appearing in the CYP21A1P gene. Among these 11 mutations, 9 are found in CAH patients and 2 created mutations were from normal individuals. Results From the DHPLC analysis using 6 fragments of amplicons, the elution profiles of the 11 mutation sites were successfully used to distinguish these common disease-causing mutations of the CYP21A2 gene. Based on this resolution, we were able to rapidly search existing sequences of mutations in the CYP21A1P gene for this malady. Conclusion DHPLC is an efficient and specific means to undertake such a program for screening patients with CAH caused by defects of the CYP21A2 gene resulting from the neighboring CYP21A1P pseudogene.

Genotype of CTX-M-type extended-spectrum beta-lactamases by denaturing high-performance liquid chromatography

Objective To investigate the genotype distribution of CTX-M-type extended-spectrum β-1actamase(ESBLs) by denaturing high-performance liquid chromatography(DHPLC) in Hunan Province and the accuracy of DHPLC assay. Methods The blaCTX-M genes of standard strains and clinical ESBLs-producing Enterobacteriaceae were amplified by multiplex PCR followed by DHPLC and genotype determination. 25 isolates randomly selected were sequenced to assess the accuracy of DHPLC method. Results Among 142 ESBLs-producing isolates, 109 isolates carried blaCTX-M gene (76. 8% ). Four different CTX-M genotypes were detected by DHPLC, including CTX-M-3 (33 isolates), CTX-M-15 (19 isolates), CTX-M-14 (52 isolates) and CTX-M-9 (5 isolates). The DHPLC typing of 25 isolates suggested that 24 isolates were verified uniformly by the sequencing, but one CTX-M-15 isolate typed by DHPLC was shown to be CTX-M-82 by sequencing. Conclusion DHPLC is a powerful tool for genotyping of the resistance gene and is worth being applied in the clinical and scientific research with accurate, rapid and economic advantages. Key words: beta-Lactamases; Escberichia coli proterins; Chromatography, high pressure liquid; Genotype

A Rapid and Accurate DHPLC Assay for Determination of Apolipoprotein E Genotypes

The variants of apolipoprotein E (apoE) are closely related to hyperlipidemia III, Alzheimer's disease (AD), coronary artery disease (CAD) and many other human lipid metabolism-related problems. A rapid and accurate genotyping method specific for polymorphisms of the APOE gene is needed for population screening as well as diagnosis of apoE-related diseases in both the research and clinical setting. A polymerase chain reaction (PCR) method was designed to generate a 191-bp amplicon, which contains two common polymorphisms located in codons 112 and 158 of exon 4 of the APOE gene. The PCR amplicons for each sample were subjected to denaturing high-performance liquid chromatography (DHPLC) analysis, which was performed under partially denaturing conditions as determined by profiling the mixture of a tested sample and a homozygous standard control amplicon at the given ratio. A total of 297 DNA samples from Chinese population were enrolled to evaluate the specificity of the assay. A blinded validation study was then performed on 130 samples randomly selected from each of the six genotype groups as determined by DHPLC profiling. The genotypes obtained with the DHPLC method were in full agreement with those obtained by direct sequencing (130/130). We have developed a PCR/DHPLC genotyping assay capable of simultaneously determining all six genotypes of APOE gene in unknown test samples at one time.

Novel mutations in the NF1 gene in Czech patients with neurofibromatosis type 1

Neurofibromatosis type 1 (NF1) is one of the most common inherited human disorders, with an estimated incidence of 1 per 3500 births. In most cases, the disease is caused either by mutation in the NF1 gene, or by a particular or complete deletion of the NF1 gene. The NF1 gene exhibits one of the highest mutation rates of any human disorder. In this experimental study of the NF1 gene, we screened the mutational spectrum of 22 unrelated patients from the Czech Republic using the denaturing high-performance liquid chromatography (DHPLC) and multiplex ligation-dependent probe amplification (MLPA) methods. We found NF1 mutations in 17 patients: 15 causal mutations were detected with the use of the DHPLC method (15/20, 75%). With the MPLA method, we also confirmed and specified two large deletions that were previously genotyped by microsatellite markers. Twelve of the above-mentioned mutations were newly found: c.1_2delATinsCC, c.1185+1G>C, c.1757_1760delCTAG, c.1642-7A>G, c.2329 T>G, c.2816delA, c.3738_3741delGTTT, c.4733 C>T, c.5220delT, c.6473_6474insGAAG, ex14_49del, ex28_49del. We present this study as a first effectual step in the routine diagnosis of the NF1 in patients from the Czech Republic.

Simultaneous detection of polymorphisms in the 3′- and 5′-UTR of thymidylate synthase gene using denaturing high-performance liquid chromatography

Polymorphism which is either in the thymidylate synthase (TS)enhancer region (TSER) of the 5-primer untranslation region (5' UTR) or in the 3-primer untranslation region (3' UTR) has been reported to be associated with the alterations in TS mRNA and protein levels. The TSER is characteristic of the presence of variable double (2R) and triple (3R) number tandem repeats (VNTRs). In addition to VNTRs, single nucleotide polymorphism (SNPs) in 3R, as well as the polymorphism of the fragment length(FLP) in the TS 3'-UTR which is characteristic of the presence or absence of a 6 bp- nucleotide fragment, has recently been reported to be associated with the response to 5-fuorouracil (5-FU)-based chemo-therapy. The aim of the present study was to develop a specifc denaturing high-performance liquid chromatography (DHPLC) method for the rapid and simultaneous detection of these variations in clinical samples. Multi-PCR primers were designed to amplify the two regions simultaneously, The 8.6 min DHPLC gradient was optimized to include the analysis of multiplexed TSER/3' UTR chromatogram peaks, allowing for the simultaneous detection of 28 bp VNTRs and 6 bp FLP under nondenaturing conditions (50). The optimal melting temperature was determined experimentally for the detection of SNP in the TSER VNTRs. Finally, the DHPLC analysis was verified in parallel with PCR-RFLP and sequencing. The optimized DHPLC method resolved 100% of the known TS variations, discriminated between homozygous and heterozy-gous genotypes. This developed DHPLC method could permit the rapid, sensitive, and accurate identification of the TS genotypes (VNTRs, SNPs, and FLP).

Identification of new single nucleotid polymorphisms (SNP) in alcohol dehydrogenase class IV ADH7 gene within a French population

Many epidemiological studies have explored the possible link between the susceptibility to alcohol related cancers, such as oesophageal cancers, and genetic variants of alcohol dehydrogenases (ADHs). Alcohol dehydrogenase class IV ADH7 is mainly expressed in the upper aero-digestive tract and is involved in the first pass ethanol metabolism. As far as we know, no study has described single nucleotide polymorphisms (SNPs) within ADH7 exons in the Caucasian population. Therefore, in a pilot study, we used the denaturing high performance liquid chromatography (DHPLC) method to screen 49 oesophageal cancer cases for SNPs in the ADH7 gene. A total of 5 SNPs was observed in this study: one SNP in the 5' non-translated regions of exon 1, two SNPs in introns 3 and 4 and two others SNPs in exons 3 and 4. The SNP located in the exon 1, which has never been described before, occurred in a reverse TATA box whereas the SNP of exon 3 was a non-silent polymorphism. Because these two SNPs could potentially affect the transcription and/or the enzyme activity, their distribution was evaluated in a representative sample of healthy Caucasians (n = 89) recruited in Lower Normandy. Frequencies of heterozygous samples ranged from 11% (exon 3) to 28% (exon 1). No homozygous samples were found. In this pilot study, the DHPLC method was suitable for both SNP screening and genotyping and allowed the detection of five SNPs in the ADH7 gene, two of which have never been described before, among the European population.

Rapid detection of glucose-6-phosphate dehydrogenase gene mutations by denaturing high-performance liquid chromatography

Objectives: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited disorder worldwide. Different kinds of G6PD mutations may result in variable severity of clinical onset in G6PD-deficient individuals. In this study, a reliable molecular diagnostic method was developed for rapid detection of G6PD gene mutation. Design and methods: Primers were designed to amplify G6PD gene fragments that were subjected to mutation screening using denaturing high-performance liquid chromatography (DHPLC) analysis. Mutations were identified by their distinct elution peak patterns and were confirmed by DNA sequencing. The assay was further validated against 29 samples from individuals with G6PD deficiency. Results: A DHPLC-based assay for G6PD mutation detection was established. The 9 common G6PD mutations in the Taiwanese and Chinese population could be distinguished through the analysis of DNA elution patterns. During the validation test with the 29 G6PD deficiency specimens, two additional rare mutations, T517C and C519G, were unveiled. Overall, the DHPLC-based mutation detection was 100% concordant with the DNA sequencing results. Conclusion: Compared to other genotyping techniques, this method requires significantly less technical time to perform and has a greatly increased throughput capacity. Hence, the DHPLC method represents a major technical advance for G6PD genotyping and should benefit G6PD-deficient individuals for proper clinical care.

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.

Simultaneous detection of variable number tandem repeats, single nucleotide polymorphisms, and allelic imbalance in the thymidylate synthase gene enhancer region using denaturing high-performance liquid chromatography

Polymorphisms in the thymidylate synthase enhancer region (TSER) have been reported to be associated with alterations in thymidylate synthase (TS) mRNA protein levels. The TSER is characterized by the presence of variable double (2R) and triple (3R) number tandem repeats (VNTRs). In addition to VNTRs, single nucleotide polymorphisms (SNPs) and allelic imbalance (AI), including loss of heterozygosity (LOH), have recently been associated with response to 5-fluorouracil (5-FU)-based chemotherapy. The aim of the current study was to develop a specific denaturing high-performance liquid chromatography (DHPLC) method for the rapid detection of these variations in the TSER in clinical samples. DHPLC analysis was validated in parallel with agarose gel electrophoresis (AGE), enzyme digestion, and quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR). The optimized DHPLC method resolved 100% of the known TSER variations, differentiated between homozygous and heterozygous genotypes, and allowed the qualitative and quantitative detection of AI, including LOH, in tumor samples. This DHPLC method was developed to permit the rapid, sensitive, and accurate identification of the TSER genotype (VNTRs, SNPs, and AI) in clinical protocols where response to flouropyrimidines may be correlated with TSER polymorphisms.

A High-Throughput Denaturing High-Performance Liquid Chromatography Method for the Identification of Variant Alleles Associated with Dihydropyrimidine Dehydrogenase Deficiency

Dihydropyrimidine dehydrogenase (DPD) is the initial, rate-limiting enzyme in the catabolism of 5-fluorouracil (5-FU). A pharmacogenetic syndrome has been described in which DPD-deficient patients are at risk for toxicity following administration of 5-FU. To date, there are at least 21 previously described mutations and/or polymorphisms that have been associated with DPD deficiency. In this study we describe the development of a highly specific, sensitive, inexpensive, and robust denaturing HPLC (DHPLC) method for rapidly identifying sequence variations (mutations and/or polymorphisms) in the gene (DPYD) that codes for the DPD enzyme. DHPLC conditions were optimized at three temperatures for analysis of the 23 exons of the DPYD gene using 25 amplicons representing the entire coding sequence, including all intron/exon boundaries (splice sites). Resolution of all 25 amplicons at the optimized temperature can be performed in 4.2 h. All 21 previously described sequence variations (mutations and/or polymorphisms) were prepared using site-directed mutagenesis from the wild-type DPYD gene, confirmed by sequence analysis, and subsequently resolved by DHPLC using the optimized conditions. These analyses generated reference chromatogram patterns for all known sequence variations previously encountered in DPD-deficient patients. In order to examine the utility and sensitivity of this approach, samples from patients with known sequence variations in the DPYD gene were analyzed. This DHPLC technique resolved 100% of the known DPYD sequence variations and differentiated between homozygous and heterozygous genotypes. We conclude that this DHPLC method is a highly specific and sensitive technique for rapidly detecting known sequence variations in the DPYD gene. In addition, this approach can be used to identify currently unrecognized unknown sequence variations in the DPYD gene and should be useful in future pharmacogenetic studies examining DPD deficiency.

DHPLC screening of cystic fibrosis gene mutations

Denaturing high performance liquid chromatography (DHPLC) using ion-pairing reverse phase chromatography (IPRPC) columns is a technique for the screening of gene mutations. In order to evaluate the potential utility of this assay method in a clinical laboratory setting, we subjected the PCR products of 73 CF patients known to bear CFTR mutations to this analytic technique. We used thermal denaturation profile parameters specified by the MELT program tool, made available by Stanford University. Using this strategy, we determined an initial analytic sensitivity of 90.4% for any of 73 known CFTR mutations. Most of the mutations not detected by DHPLC under these conditions are alpha-substitutions. This information may eventually help to improve the MELT algorithm. Increasing column denaturation temperatures for one or two degrees above those recommended by the MELT program allowed 100% detection of CFTR mutations tested. By comparing DHPLC methodology used in this study with the recently reported study based on Wavemaker 3.4.4 software (Transgenomic, Omaha, NE) [Le Marechal et al., 2001) and with previous SSCP analysis of CFTR mutations [Ravnik-Glavac et al., 1994] we emphasized differences and similarities in order to refine the DHPLC system and discuss the relationship to the alternative approaches. We conclude that the DHPLC method, under optimized conditions, is highly accurate, rapid, and efficient in detecting mutations in the CFTR gene and may find high utility in screening individuals for CFTR mutations. Hum Mutat 19:374-383, 2002. Published 2002 Wiley-Liss, Inc.

Slowly but surely towards better scanning for mutations

3065, Aumum. As the number of dii genes described and the number of candi- date genes for diseases increases, so does the need for better DNA scan- ning methods. Notionally, the search for unknown mutations in DNA should be simple, thus, surely all one needs to do is to sequence the DNA. The reason why this does not appear to be true is complex, but perhaps the three major reasons are: (1) that the number of laboratories in the world who are expert sequencers is limited; (2) that many applications call for the analysis of hundreds of disease genes; and (3) that precise sequencing is time-consuming and expensive. These reasons have led to the development and refinement of a large number of DNA scanning methods in an attempt to develop economical and effective methods. There have been numerous articles reviewing and describing such meth- ods in recent years’-7. This work has been moving towards the elusive ‘ideal scanning method’ (Box 1). In the last year or so, there have been no dramatic novel approaches described, except perhaps the DHPLC (denaturing high-performance liquid chromatography) method, a hetero- duplex variant (see below), but there have been signiticant studies with, or advances made in, the methods described so far, and these advances are the subject of this article. An earlier issue of Trends