Multiplex Single Base Extension Research Articles

Population genetic variation of the Southern Ocean krill, Euphausia superba, in the Western Antarctic Peninsula region based on mitochondrial single nucleotide polymorphisms (SNPs)

The Southern Ocean krill, Euphausia superba, is one of the best-studied marine zooplankton species in terms of population genetic diversity and structure; with few exceptions, previous studies have shown the species to be genetically homogeneous at larger spatial scales. The goals of this study are to examine sub-regional scale population genetic diversity and structure of E. superba using molecular characters selected with this goal in mind, and to thereby examine hypotheses of the source(s) of recruitment for krill populations of the Western Antarctic Peninsula (WAP). Collections were made throughout the WAP region during US GLOBEC cruises in austral fall, 2001 and 2002. A total of 585 E. superba (including all 6 furcilia larval stages, juveniles, and adults) was analyzed after confirmation of species identification using a competitive multiplexed species-specific PCR (SS-PCR) reaction based on mitochondrial cytochrome oxidase I (mtCOI) sequences. The molecular markers used were allele frequencies at single nucleotide polymorphism (SNP) sites in the gene encoding mitochondrial Cytochrome b (cyt b). Four SNP sites that showed desirable patterns of allelic variation were selected; alleles were detected using a multiplexed single-base extension PCR protocol. A total of 22 SNP haplotypes (i.e., strings of polymorphisms at the four SNP sites) was observed; haplotype diversity (Hd)=0.811 (s.d.=0.008). Analysis of molecular variation within and among samples, areas (i.e., Marguerite Bay, Crystal Sound, shelf, and offshore) and collection years revealed no difference between 2001 and 2002 collections overall, although differences between 2001 and 2002 collections from Marguerite Bay explained 7.4% of the variance ( F ST=0.072; p=0.002±0.001). Most of the variation (96.3%) occurred within samples each year, with no significant differentiation among areas. There was small, but significant differentiation among samples within areas in 2001 (4.6%; F ST=0.045; p=0.015±0.003) and 2002 (6.3%; F ST=0.062; p=0.000±0.000). There was evidence of life stage-specific spatial differentiation for furcilia in 2002 for F1 (18.1%, p=0.000±0.000) and F2 (9.2%, p=0.001±0.001). The significant differentiation among samples for E. superba within areas was interpreted as evidence of multiple sources of recruitment of E. superba in the WAP region, consistent with advective transport in observed circulation patterns and reproduction and spawning in both offshore and shelf habitats. Further population genetic analysis at sub-regional scales is needed to understand and eventually predict population dynamic processes (e.g., recruitment, migration, retention, and over-wintering) of Southern Ocean krill.

Robust CYP2D6 genotype assay including copy number variation using multiplex single-base extension for Asian populations

Background We developed a CYP2D6 genotyping method that includes copy number variation (CNV) and recently known functional haplotypes using multiplex single-base extension (SBE). Methods Twelve CYP2D6 alleles ( *1, *2, *5, *10, *14, *18, *21, *41, *49, *52, *60, and a duplication of CYP2D6) were genotyped using 2 PCR reactions followed by multiplex SBE with 10 primers and singleplex SBE with 1 primer. The result from 758 Korean samples was validated by comparison with the results of direct sequencing or other genotyping methods. We also genotyped 89 Chinese and 122 Vietnamese subjects to determine the presence of recently identified functional alleles. Results All 12 CYP2D6 alleles, including gene deletion and duplication, were obviously discriminated. The concordance rate was 100% between our method and other methods. Our method also covered over 98% of the CYP2D6 genotypes in Japanese and Chinese subjects based on reported data. In addition to published genotypes, *14, *21, *41, *49, and *52 were found in about 5% in Chinese and Vietnamese. Conclusions The CYP2D6 genotyping method may be clinically applicable for Asian populations. The method can be improved easily to cover other ethnic groups by utilizing additional haplotype tagging SNPs.

Association of RS2200733 but Not RS10033464 on 4q25 with Atrial Fibrillation Based on the Recessive Model in a Taiwanese Population

Objectives: To determine the association between genetic variants on chromosome 4q25 and atrial fibrillation (AF) in a Taiwanese population. Methods: We enrolled 200 patients with AF (mean age: 67 ± 13 years) and 158 controls (mean age: 63 ± 10 years). The genotypes of five SNPs, RS2634073, RS2200733, RS13143308, RS2220427 and RS10033464, were determined using multiplex single base extension methods. Results: The distribution of the RS2200733 and RS10033464 genotypes did not significantly deviate from the Hardy-Weinberg equilibrium in the control group. The distribution of the RS2200733 genotypes differed significantly between the AF group and the controls (p = 0.03), whereas the distribution of the RS10033464 genotypes did not (p = 0.49). At RS2200733, patients with the CC genotype exhibited a 0.45 times higher risk of developing AF than those with the TT genotype (p = 0.02) and a recessive model was suggested (p = 0.01). After adjusting for various covariates, patients with the CC genotype remained recessively associated with a lower risk of developing AF than those with the TT genotype (odds ratio: 0.27, 95% confidence interval: 0.11–0.65; p < 0.01). Conclusions: In the Taiwanese, there is an association between SNP RS2200733 – but not RS10033464 – and the development of AF. Based on a recessive model of inheritance, individuals with SNP RS2200733 genotype CC are at a lesser risk of developing AF.

A genetic investigation of Korean mummies from the Joseon Dynasty

Two Korean mummies (Danwoong-mirra and Yoon-mirra) found in medieval tombs in the central region of the Korean peninsula were genetically investigated by analysis of mitochondrial DNA (mtDNA), Y-chromosomal short tandem repeat (Y-STR) and the ABO gene. Danwoong-mirra is a male child mummy and Yoon-mirra is a pregnant female mummy, dating back about 550 and 450 years, respectively. DNA was extracted from soft tissues or bones. mtDNA, Y-STR and the ABO gene were amplified using a small size amplicon strategy and were analyzed according to the criteria of ancient DNA analysis to ensure that authentic DNA typing results were obtained from these ancient samples. Analysis of mtDNA hypervariable region sequence and coding region single nucleotide polymorphism (SNP) information revealed that Danwoong-mirra and Yoon-mirra belong to the East Asian mtDNA haplogroups D4 and M7c, respectively. The Y-STRs were analyzed in the male child mummy (Danwoong-mirra) using the AmpFlSTR® Yfiler PCR Amplification Kit and an in-house Y-miniplex plus system, and could be characterized in 4 loci with small amplicon size. The analysis of ABO gene SNPs using multiplex single base extension methods revealed that the ABO blood types of Danwoong-mirra and Yoon-mirra are AO01 and AB, respectively. The small size amplicon strategy and the authentication process in the present study will be effectively applicable to future genetic analyses of various forensic and ancient samples.

Simple, Efficient, and Cost-Effective Multiplex Genotyping with Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry of Hemoglobin Beta Gene Mutations

A number of common mutations in the hemoglobin beta (HBB) gene cause beta-thalassemia, a monogenic disease with high prevalence in certain ethnic groups. As there are 30 HBB variants that cover more than 99.5% of HBB mutant alleles in the Thai population, an efficient and cost-effective screening method is required. Three panels of multiplex primer extensions, followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were developed. The first panel simultaneously detected 21 of the most common HBB mutations, while the second panel screened nine additional mutations, plus seven of the first panel for confirmation; the third panel was used to confirm three HBB mutations, yielding a 9-Da mass difference that could not be clearly distinguished by the previous two panels. The protocol was both standardized using 40 samples of known genotypes and subsequently validated in 162 blind samples with 27 different genotypes (including a normal control), comprising heterozygous, compound heterozygous, and homozygous beta-thalassemia. Results were in complete agreement with those from the genotyping results, conducted using three different methods overall. The method developed here permitted the detection of mutations missed using a single genotyping procedure. The procedure should serve as the method of choice for HBB genotyping due to its accuracy, sensitivity, and cost-effectiveness, and can be applied to studies of other gene variants that are potential disease biomarkers.

Simultaneous genotyping of 11 non-synonymous SNPs in the 4 glutathione peroxidase genes using the multiplex single base extension method

Background Glutathione peroxidase (Gpx) is one of the major antioxidant enzymes involved in scavenging hydrogen peroxide and lipid hydroperoxides produced during normal metabolism or after oxidative insult. In the Gpx genes, a number of single nucleotide polymorphisms (SNPs) have been identified and their associations with various diseases have been reported. In the present study, we used a multiplex single base extension (MSBE) technique to genotype multiple non-synonymous SNPs in the Gpx1, Gpx2 , Gpx3 and Gpx4 genes simultaneously. Methods Seven templates for the MSBE reaction, involving 11 SNPs corresponding to non-synonymous mutations were amplified by multiplex PCR. Availability of the MSBE method was validated by genotyping DNA from 91 Japanese, 91 German and 93 Xhosa healthy subjects. Results A simple and reproducible method for simultaneous genotyping of multiple SNPs in the Gpx genes was established. Of the 11 SNPs, only Gpx1 P200L was polymorphic in all three ethnic groups and the genotype distributions differed significantly among the three populations. On the other hand, little heterogeneity was observed for Gpx2 R146C, Gpx2 P126L, Gpx1 A194T and Gpx4 S2N, and no heterogeneity was observed for Gpx1 L6P, Gpx1 R5P, Gpx3 F128L, Gpx3 K144X, Gpx4 A88V and Gpx4 S227L. Conclusion The MSBE procedure described here was proven to be applicable for population studies. Application of this method will provide comprehensive information for studying the relationship between SNPs in the Gpx genes and risks for various diseases.

Multiplex single base extension method for simultaneous genotyping of non‐synonymous SNP in the three human SOD genes

The superoxide dismutases (SOD) are a family of enzymes that function as the first line of antioxidant defense against highly reactive superoxide radicals. In SOD genes, a number of SNP have been identified and their associations with various diseases have been reported. In the present study, we applied a multiplex single base extension technique to genotype multiple non-synonymous SNP in the SOD1, SOD2 and SOD3 genes simultaneously, and examined allele distributions in healthy Caucasian (German), Asian (Japanese) and African (Xhosa) populations. Of the ten SNP investigated, two (SOD2 Ala16Val, SOD3 Ala58Thr) were polymorphic in all three ethnic groups and the genotype distributions showed significant inter-group differences. On the other hand, a small number of heterozygotes were observed for three SNP (SOD2 Ser10Ile, SOD3 Ala91Thr, SOD3 Arg231Gly) and no heterogeneity was observed for the remaining five (SOD1 Thr40Ile, SOD1 Asn87Ser, SOD2 Arg156Trp, SOD2 Gly76Arg, SOD2 Glu66Val). Analyses of associations between SOD genotypes and levels of plasma SOD activity demonstrated that SOD2 Ala16Val, a dimorphism leading to substitution in the mitochondrial targeting sequence of SOD2, significantly influences plasma SOD2 activity, and that SOD3 Arg231Gly, leading to substitution in the heparin-binding domain of SOD3, significantly influences plasma total SOD activity.

Determination of cis/trans phase of variations in the MC1R gene with allele‐specific PCR and single base extension

The MC1R gene encodes a protein with key regulatory functions in the melanin synthesis. A multiplex PCR and a multiplex single base extension protocol were established for genotyping six exonic MC1R variations highly penetrant for red hair (R), four exonic MC1R variations weakly penetrant for red hair (r), two frameshift variations highly penetrant for red hair (R) and three variations in the promoter region. We genotyped 600 individuals from Denmark using either CE or MALDI-TOF MS as the detection platform. A total of 62 individuals were genotyped R/R and among the 62 individuals, 57 had red hair and five had blond hair colour. Two different R alleles may be located in cis (RR/-) position or trans (R/R) position, and the phenotype associated with RR/- and R/R may be different. Two allele-specific PCRs were established with primers targeting the -G445A variation in the MC1R promoter and the allele-specific PCR products were used in the multiplex single base extension assay. In all 62 individuals, the MC1R variants were situated in trans position. Another 18 individuals with red hair colour were either genotyped R/- or R/r, suggesting that other genes influence hair colour.

Multiplex single-base extension typing to identify nuclear genes required for RNA editing in plant organelles

We developed a multiplex single-base extension single-nucleotide polymorphism-typing procedure for screening large numbers of plants for mutations in mitochondrial RNA editing. The high sensitivity of the approach detects changes in the RNA editing status generated in total cellular cDNA from pooled RNA preparations of up to 50 green plants. The method has been employed to tag several nuclear encoded genes required for RNA editing at specific sites in mitochondria of Arabidopsis thaliana. This approach will allow large-scale screening for mutations in genes encoding trans-factors for many types of RNA editing as well as for other RNA modifications.

The Mediterranean basin: A population genetic study based on 25 X-chromosome SNPs

The X-chromosome has valuable characteristics for population genetic studies. In order to investigate the genetics of the human Mediterranean populations further, we developed a 25 X-chromosome SNP-multiplex typing system. The system was based on PCR multiplex amplification and subsequent multiplex single base extension with the SNaPshot reaction, capillary electrophoresis and multicolor fluorescence detection. We investigated 11 Mediterranean populations with the 25 X-chromosome SNPs. A high overall homogeneity was found among the Mediterranean populations except for Moroccans, who differed genetically from the rest of the populations in the Mediterranean area. This result supports the hypothesis of a low incidence of the south–north genetic interchange at the western shores of the Mediterranean basin. A low genetic distance was found between populations in the Middle East and the western part of the Mediterranean area most likely reflecting the strong effect of the Neolithic wave. A certain level of background linkage disequilibrium among the 25 SNPs on the X-chromosome in Ibiza and Cosenza was observed, possibly as a consequence of their demographic history.

Development and evaluation of a generic tag array to detect and genotype noroviruses in water

Noroviruses are the leading cause of nonbacterial gastroenteritis outbreaks in the United States, some of which are caused by the ingestion of contaminated water. Detection and genotypic characterization of noroviruses is commonly performed by reverse transcription-polymerase chain reaction (RT-PCR) followed by sequencing. However, sequencing of products amplified from environmental water samples is often hindered by the co-amplification of non-specific cDNA. To overcome this issue, a generic microarray was evaluated to genotype noroviruses by probe hybridization. RT-PCR amplicons are used in a single base extension (SBE) reaction where genotype-specific probes are labeled and then hybridized to an Affymetrix GeneChip® GenFlex™ Tag Array for detection. Using a standardized, multiplex SBE reaction, genotyping of representative strains was accomplished through the identification of genotype-specific patterns of positive hybridization results. Furthermore, the SBE-GenFlex array method was successful in the genotype identification of noroviruses seeded into tap and Ohio River water samples. This study shows the utility of using a microarray to genotype noroviruses in complex environmental matrices.

Microarray-based detection of Korean-specific BRCA1 mutations

A reliable multiplex assay procedure to detect human genetic mutations in the breast cancer susceptibility gene BRCA1 using zip-code microarrays and single base extension (SBE) reactions is described. Multiplex PCR amplification was performed to amplify the genomic regions containing the mutation sites. The PCR products were then employed as templates in subsequent multiplex SBE reactions using bifunctional primers carrying a unique complementary zip sequence in addition to a mutation-site-specific sequence. The SBE primers, terminating one base before their mutation sites, were extended by a single base at a mutation site with a corresponding biotin-labeled ddNTP. Hybridization of the SBE products to zip-code microarrays was followed by staining with streptavidin-Cy3, leading to successful genotyping of several selected BRCA1 mutation sites with wild-type and heterozygote mutant samples from breast cancer patients. This work has led to the development of a reliable DNA microarray-based system for the diagnosis of human genetic mutations.

Novel CYP2C9 Promoter Variants and Assessment of Their Impact on Gene Expression

There are a considerable number of reports identifying and characterizing genetic variants within the CYP2C9 coding region. Much less is known about polymorphic promoter sequences that also might contribute to interindividual differences in CYP2C9 expression. To address this problem, approximately 10,000 base pairs of CYP2C9 upstream information were resequenced using 24 DNA samples from the Coriell Polymorphism Discovery Resource. Thirty-one single-nucleotide polymorphisms (SNPs) were identified; nine SNPs were novel, whereas 22 were reported previously. Using both sequencing and multiplex single-base extension, individual SNP frequencies were determined in 193 DNA samples obtained from unrelated, self-reported Hispanic Americans of Mexican descent, and they were compared with similar data obtained from a non-Latino white cohort. Significant interethnic differences were observed in several SNP frequencies, some of which seemed unique to the Hispanic population. Analysis using PHASE 2.1 inferred nine common (>1%) variant haplotypes, two of which included the g.3608C>T (R144C) CYP2C9(*)2 and two the g.42614A>C (I359L) CYP2C9(*)3 SNPs. Haplotype variants were introduced into a CYP2C9/luciferase reporter plasmid using site-directed mutagenesis, and the impact of the variants on promoter activity assessed by transient expression in HepG2 cells. Both constitutive and pregnane X receptor-mediated inducible activities were measured. Haplotypes 1B, 3A, and 3B each exhibited a 65% decrease in constitutive promoter activity relative to the reference haplotype. Haplotypes 1D and 3B exhibited a 50% decrease and a 40% increase in induced promoter activity, respectively. These data suggest that genetic variation within CYP2C9 regulatory sequences is likely to contribute to differences in CYP2C9 phenotype both within and among different populations.

Identification of West Eurasian mitochondrial haplogroups by mtDNA SNP screening: Results of the 2006–2007 EDNAP collaborative exercise

The European DNA Profiling (EDNAP) Group performed a collaborative exercise on a mitochondrial (mt) DNA screening assay that targeted 16 nucleotide positions in the coding region and allowed for the discrimination of major west Eurasian mtDNA haplogroups. The purpose of the exercise was to evaluate the stability and reproducibility of the self-developed multiplex-PCR and multiplex-single base extension kit by blind-testing saliva and hair shaft samples provided by the organizing laboratory. The overall success rate in obtaining useful results was high given that some of the participating laboratories had no previous experience with the technology and/or mtDNA analysis. The results of this collaborative exercise stimulate the expansion of screening methods in forensic laboratories to increase efficiency and performance of mtDNA typing, and thus demonstrates that mtDNA SNP typing is a powerful tool for forensic casework analysis.

SIMULTANEOUS GENOTYPING OF CYP2D6*3, *4, *5 AND *6 POLYMORPHISMS IN A SPANISH POPULATION THROUGH MULTIPLEX LONG POLYMERASE CHAIN REACTION AND MINISEQUENCING MULTIPLEX SINGLE BASE EXTENSION ANALYSIS

1. The aim of the present study was to perform a descriptive study of the prevalence of the four major CYP2D6 poor metaboliser (PM) alleles (*3, *4, *5 and *6) in a Spanish population (n = 290) using a method based on a new combination of multiplex long polymerase chain reaction (PCR) and minisequencing through multiplex single base extension (SBE) analysis. 2. The method was validated using different strategies, such as allelic discrimination assay and PCR-restriction fragment length polymorphism (RFLP). 3. The allele frequencies were similar to those described for other Spanish populations, namely 0.9% (95% confidence interval (CI) 0.5-1.3), 16.4% (95% CI 14.9-18.0), 2.7% (95% CI 2.0-3.4) and 0.7% (95% CI 0.3-1.0) for the *3, *4, *5 and *6 alleles, respectively. The results were satisfactory and left little doubt as to the genotypes, which were confirmed either by allelic discrimination assay (*4 and *6) or PCR-RFLP (*3) with 100% concordance. 4. The present study corroborates the low prevalence of the most frequent polymorphism (CYP2D6*4) that leads to null CYP2D6 activity in Spain and the allelic geographical gradient between Caucasian populations in the north and south. The present study reports a technique for the detection of four polymorphisms that account for 98% of the CYP2D6 defect alleles. This multiplex long PCR-SBE technique is a combination of several known methods to genotype CYP2D6 alleles (*3, *4, *5 and*6). Given the importance of CYP2D6 in drug metabolism and the need to genotype a large number of samples, we believe that this method will find broad application.

Multiplexed p53 Mutation Detection by Free-Solution Conjugate Microchannel Electrophoresis with Polyamide Drag-Tags

We report a new, bioconjugate approach to performing highly multiplexed single-base extension (SBE) assays, which we demonstrate by genotyping a large panel of point mutants in exons 5-9 of the p53 gene. A series of monodisperse polyamide "drag-tags" was created using both chemical and biological synthesis and used to achieve the high-resolution separation of genotyping reaction products by microchannel electrophoresis without a polymeric sieving matrix. A highly multiplexed SBE reaction was performed in which 16 unique drag-tagged primers simultaneously probe 16 p53 gene loci, with an abbreviated thermal cycling protocol of only 9 min. The drag-tagged SBE products were rapidly separated by free-solution conjugate electrophoresis (FSCE) in both capillaries and microfluidic chips with genotyping accuracy in excess of 96%. The separation requires less than 70 s in a glass microfluidic chip, or about 20 min in a commercial capillary array sequencing instrument. Compared to gel electrophoresis, FSCE offers greater freedom in the design of SBE primers by essentially decoupling the length of the primer and the electrophoretic mobility of the genotyping products. FSCE also presents new possibilities for the facile implementation of SBE on integrated microfluidic electrophoresis devices for rapid, high-throughput genetic mutation detection or SNP scoring.

Molecular Identification of "Chuanxiong" by Nucleotide Sequence and Multiplex Single Base Extension Analysis on Chloroplast trnK Gene

Chloroplast trnK gene sequences of Cnidium officinale and Ligusticum chuanxiong were determined to establish an effective method for identifying Japanese Senkyu and Chinese Chuanxiong, the two which have the same drug name in Chinese characters, similar external feature, but different botanical origins. Three sites of nucleotide differences were found between these 2 species at positions 767,924 and 964 from upstream in trnK gene sequence, allowing molecular identification of the two plants and crude drugs. Further, three kinds of specific primers of 14 mer, 23 mer and 30 mer long were designed to detect these 3 sites of marker nucleotides. By using multiplex single base extension (MSBE) analysis with the 3 specific primers, C. officinale and L. chuanxiong could be distinguished clearly by the electrophoretograms, where 3 peaks with different color of ddTMP, ddCMP and ddTMP were observed in case of C. officinale and those of ddGMP, ddAMP and ddGMP in L. chuanxiong. Moreover, trnK gene sequence of "Dongxiong," a kind of Chuanxiong cultivated in Northeast China, suggested that its botanical origin was C. officinale.

Simultaneous genotyping of CYP2C9*2, *3, and 5′ flanking region (C-1189T) polymorphisms in a Spanish population through a new minisequencing multiplex single-base extension analysis

To conduct a descriptive study on the prevalence of relevant cytochrome P450 2C9 (CYP2C9) polymorphisms--the *2, *3, and 5' flanking region (C-1189T)--in a Spanish population using a new minisequencing fluorescent method through a multiplex single base extension (SBE) analysis. The method simultaneously and accurately genotypes the CYP2C9 polymorphisms studied and is available as a commercial protocol (SNaPshot). Various strategies, including restriction fragment length polymorphism (RFLP) and Taqman, were used to validate the methodology. The frequencies of alleles CYP2C9*2 (12%) and *3 (6.2%) were similar to those described for other Caucasian populations. The frequency of allele t at the 5' flanking region was 62%, which is close to the percentage reported in Japanese and French populations. The four haplotypes inferred in our samples and their frequencies were consistent with those reported in other studies. Our results confirm previously reported Caucasian frequencies for the CYP2C9*2 and *3 alleles and, for the first time, provide data on the frequency of the CYP2C9 5' flanking region (C-1189T), a recently described polymorphism, in a Spanish population. The SBE technique detects unequivocally the three polymorphisms in a single reaction, which makes it suitable for the analysis of CYP2C9 in the many therapeutic situations in which it is involved.

Diagnosis of HNF-1 mutations on a PNA zip-code microarray by single base extension

In the present study, we exploited the superior features of peptide nucleic acids (PNAs) to develop an efficient PNA zip-code microarray for the detection of hepatocyte nuclear factor-1α (HNF-1α) mutations that cause type 3 maturity onset diabetes of the young (MODY). A multi-epoxy linker compound was synthesized and used to achieve an efficient covalent linking of amine-modified PNA to an aminated glass surface. PCR was performed to amplify the genomic regions containing the mutation sites. The PCR products were then employed as templates in a subsequent multiplex single base extension reaction using chimeric primers with 3′ complementarity to the specific mutation site and 5′ complementarity to the respective PNA zip-code sequence on the microarray. The primers were extended by a single base at each corresponding mutation site in the presence of biotin-labeled ddNTPs, and the products were hybridized to the PNA microarray. Compared to the corresponding DNA, the PNA zip-code sequence showed a much higher duplex specificity for the complementary DNA sequence. The PNA zip-code microarray was finally stained with streptavidin-R-phycoerythrin to generate a fluorescent signal. Using this strategy, we were able to correctly diagnose several mutation sites in exon 2 of HNF-1α with a wild-type and mutant samples including a MODY3 patient. This work represents one of the few successful applications of PNA in DNA chip technology.

Simultaneous Genotyping of Nine Polymorphisms in Xenobiotic-Metabolizing Enzymes by Multiplex PCR Amplification and Single Base Extension

Studies have reported a large interindividual variation in susceptibility to health effects caused by exposure to xenobiotic compounds such as drugs or chemical carcinogens. There is evidence that this can be partly explained by the existence of genetic polymorphisms in metabolic enzymes, such as cytochrome P450 (CYP450), N -acetyltransferases (NATs), and glutathione S -transferases (GSTM, GSTT, GSTP) [see, e.g., Refs. (1)(2)(3)(4)]. However, studies investigating associations between genetic polymorphisms and disease have reported conflicting results, probably caused by insufficient statistical power (5). Moreover, the majority of these studies focused on single polymorphisms. Regarding the number of genes implicated in the metabolism of xenobiotics and the large number of polymorphisms present in the human genome (6), these approaches fail to fully determine the role of genetic variation in an individual’s susceptibility to xenobiotic exposures. Such observations underline the need for methodologies that allow for high-throughput, low-cost genotyping of multiple polymorphisms in large populations (7)(8). In this study we describe the development, validation, and application of a cost-effective and rapid method for simultaneous genotyping of nine polymorphisms in five key enzymes involved in metabolism of xenobiotics: CYP1A2, GSTM1, GSTP1, GSTT1, and NAT2. The fragments containing the nine single-nucleotide polymorphisms (SNPs) were amplified in one sevenplex and one duplex PCR reaction (Table 1⇓ ). Primers were obtained from Qiagen. For the sevenplex PCR, a 50-μL reaction mixture was prepared containing PCR buffer, 0.2 mM deoxynucleotide triphosphates, 0.5 mM MgCl2, 1.25 U of Platinum® Taq Polymerase (Invitrogen), and 200 ng of template DNA. The final primer concentrations were 0.22 μM (for GSTP1 * 3 , GSTT1 , NAT2 * 6 , and NAT2 * 7 ), 0.45 μM (for CYP1A2 * 1F and NAT2 * 5 ), and 0.16 μM (for GSTP1 * 2 ). PCR was …