Genotyping of exons 1 to 20 in Duchenne muscular dystrophy by universal multiplex PCR and short‐end capillary electrophoresis

One rapid CE method was established to diagnose Duchenne muscular dystrophy (DMD). DMD is a severe recessive inherited disorder frequently caused by gene deletions. Among them, exons 1–20 account for nearly 30% of occurrences. In this study, the universal multiplex PCR was used to enhance the fluorescently labeling efficiency, which was performed only by one universal fluorescent primer. After PCR, a short‐end injection CE (short‐end CE) speeded up the genotyping of the DMD gene. This method involved no extra purification, and was completed within 9 min. The CE conditions contained a polymer solution of 1.5% hydroxylethylcellulose in 1× TBE buffer at 6 kV for separation. This method was applied to test six DMD patients and one healthy male person. The results showed good agreement with those of multiplex ligation‐dependent probe amplification. This method can be applied for clinical diagnosis of DMD disease. Accurate diagnosis of the DMD gene is the best way to prevent the disease.     

Identification of deletion and duplication genotypes of the PMP22 gene using PCR-RFLP, competitive multiplex PCR, and multiplex ligation-dependent probe amplification: a comparison

We evaluated the efficacy of PCR-RFLP, competitive multiplex PCR, and a commercially available system of multiplex ligation-dependent probe amplification (MLPA) for the determination of deletion and duplication genotypes of the PMP22 gene. We compared the methods for efficiency, sensitivity, and specificity. We determined the gene dosage of the PMP22 gene via PCR-RFLP, competitive multiplex PCR, and MLPA. To demonstrate the sensitivity and accuracy of these three methods, a total of 185 samples from 42 patients with hereditary neuropathy with liability to pressure palsies (HNPP), 57 patients with Charcot-Marie-Tooth disease type 1A (CMT1A), and 86 unaffected individuals, were analyzed. Molecular diagnosis by PCR-RFLP was performed on all 185 samples; 24 HNPP deletions and 33 CMT1A duplications were identified. In contrast, 25 HNPP deletions and 38 CMT1A duplications were identified correctly using competitive multiplex PCR and MLPA. Six samples were incorrectly identified by PCR-RFLP (one HNPP deletion and five CMT1A duplications). Competitive multiplex PCR and MLPA demonstrated reliability and relative speed compared to PCR-RFLP; they were superior to PCR-RFLP for gene dosage quantification. Multiplex PCR and MLPA should be the methods of choice for detection of deletion and duplication genotypes in molecular genetic diagnoses.     

Quantitative polymerase chain reaction and microchip electrophoresis to detect major rearrangements of the low-density lipoprotein receptor gene causing familial hypercholesterolemia

A variety of rearrangements in the low-density lipoprotein receptor (LDLR) gene cause severe forms of familial hypercholesterolemia (FH). However, current methods for searching these abnormalities in FH samples, e.g., Southern and Northern Blot, are labor-intensive and not routinely used by diagnostic laboratories. We developed a simpler approach based on the quantitative polymerase chain reaction (PCR) amplification of part or all gene's coding sequences by a series of multiplex amplifications comprising three nonadjacent gene sections plus a fourth section used as an internal reference. Thereafter, the analysis of these PCR products by microchip electrophoresis revealed either deletions or duplications in the investigated gene sections through the simple comparison of electropherograms obtained from mutant and control samples. This required primers leading to well-resolved peaks with minimal size differences among coamplified products and PCR conditions allowing a linear quantitative response to template amount variations as those caused by duplication or deletion of specific gene sections. Also, the inclusion of exon 17 amplification product as an internal reference in each multiplex PCR allowed the normalization of quantitative results by dividing the area of each amplified section by the area of exon 17. The comparison of these ratios calculated from 10 carriers of 6 LDLR known rearrangements with those obtained from 14 control samples showed that gross deletions roughly halved and duplications doubled the ratio values of exons involved in the mutation. This allowed to distinguish gross mutations from sample-to-sample differences that reached at maximum 8% variation over mean values.     

Mutation analysis of PAH gene and characterization of a recurrent deletion mutation in Korean patients with phenylketonuria

Phenylketonuria (PKU; MIM 261600) is an autosomal recessive metabolic disorder caused by a deficiency of phenylalanine hydroxylase (PAH; EC 1.14.16.1). Point mutations in the PAH gene are known to cause PKU in various ethnic groups, and large deletions or duplications account for up to 3% of the PAH mutations in some ethnic groups. However, a previous study could not identify approximately 14% of the mutant alleles by sequence analysis in Korean patients with PKU, which suggests that large deletions or duplication might be frequent causes of PKU in Koreans. To test this hypothesis, we performed multiplex ligation-dependent probe amplification (MLPA) for the identification of uncharacterized mutant alleles after PAH sequence analysis of 33 unrelated Korean patients with PKU. Bi-directional sequencing of the PAH exons and flanking intronic regions revealed 27 different mutations, including four novel mutations (two missense and two deletion mutations), comprising 57/66 (86%) mutant alleles. MLPA identified a large deletion that encompassed exons 5 and 6 in four patients, another large deletion that extended from exon 4 to exon 7 in one patient, and a duplication of exon 4 in one patient. Chromosomal walking characterized the deletion breakpoint of the most common large deletion that involved exons 5 and 6 (c.456_706+138del). The present study shows that the allelic frequency of exon deletion or duplication is 9% (6/66) in Korean PKU patients, which suggests that these mutations may be frequent causes of PKU in Korean subjects.     

Developments toward a complete micro-total analysis system for Duchenne muscular dystrophy diagnosis

The diagnosis of Duchenne muscular dystrophy (DMD) has historically utilized either PCR or requires Southern blot analysis, a southern blot analysis, however, is not amenable to incorporation in a microdevice format. A PCR amplification-based method has been developed, and we have previously coupled this amplification with microchip separation of the PCR fragments for DMD diagnosis. Diagnoses of affected patients were performed by comparing exon concentrations to those of control samples amplified at the same time. To accurately identify mutations in patient samples, this work established normal ranges for the concentration of each amplified exon fragment using control samples amplified over successive days. Our studies show that the number of cycles used in the amplification process affects this range. Affected patient samples were analyzed using these normal ranges and the mutations detected by Southern blot analysis were also diagnosed using the microchip separation method.

Employing the microchip separation method decreases the time required for the analysis, but the time required for DNA purification and PCR amplification must also be decreased for faster total analysis of patient samples. Development of microchip methods for these processing steps is one approach for reducing the individual times, while also providing the possibility of integrating these steps in a single device. Here we report on the microchip extraction of genomic DNA from whole blood using a novel sol–gel matrix that is easily formed in microdevices. IR-mediated PCR amplification of a β-globin fragment from genomic DNA followed by electrophoretic analysis on a single integrated microdevice is presented for the first time. Work towards the development of a micro-total analysis device for DMD diagnosis, through integration of all processing steps on a single device, is also discussed.     

Exon deletions of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemics

A consistent finding of many studies describing the spectrum of mutant phenylalanine hydroxylase (PAH) alleles underlying hyperphenylalaninemia is the impossibility of achieving a 100% mutation ascertainment rate using conventional gene-scanning methods. These methods include denaturing gradient gel electrophoresis (DGGE), denaturing high performance liquid chromatography (DHPLC), and direct sequencing. In recent years, it has been shown that a significant proportion of undetermined alleles consist of large deletions overlapping one or more exons. These deletions have been difficult to detect in compound heterozygotes using gene-scanning methods due to a masking effect of the non-deleted allele. To date, no systematic search has been carried out for such exon deletions in Italian patients with phenylketonuria or mild hyperphenylalaninemia. We used multiplex ligation- dependent probe amplification (MLPA), comparative multiplex dosage analysis (CMDA), and real-time PCR to search for both large deletions and duplications of the phenylalanine hydroxylase gene in Italian hyperphenylalaninemia patients. Four deletions removing different phenylalanine hydroxylase (PAH) gene exons were identified in 12 patients. Two of these deletions involving exons 4-5-6-7-8 (systematic name c.353-?_912 + ?del) and exon 6 (systematic name c.510-?_706 + ?del) have not been reported previously. In this study, we show that exon deletion of the PAH gene accounts for 1.7% of all mutant PAH alleles in Italian hyperphenylalaninemics.     

Precise characterization method of antibody‐conjugated magnetic nanoparticles for pathogen detection using stuffer‐free multiplex ligation‐dependent probe amplification

Antibody‐conjugated magnetic nanoparticles (Ab‐MNPs) have potential in pathogen detection because they allow target cells to be easily separated from complex sample matrices. However, the sensitivity and specificity of pathogen capture by Ab‐MNPs generally vary according to the types of MNPs, antibodies, and sample matrices, as well as preparation methods, including immobilization. Therefore, achieving a reproducible analysis utilizing Ab‐MNPs as a pathogen detection method requires accurate characterization of Ab‐MNP capture ability and standardization of all handling processes. In this study, we used high‐resolution CE‐single strand conformational polymorphism coupled with a stuffer‐free multiplex ligation‐dependent probe amplification system to characterize Ab‐MNPs. The capture ability of Ab‐MNPs targeting Salmonella enteritidis and nine pathogens, including S. enteritidis, was analyzed in phosphate buffer and milk. The effect of storage conditions on the stability of Ab‐MNPs was also assessed. The results showed that the stuffer‐free multiplex ligation‐dependent probe amplification system has the potential to serve as a standard characterization method for Ab‐MNPs. Moreover, the precise characterization of Ab‐MNPs facilitated robust pathogen detection in various applications.     

连接反应介导的等位基因特异性扩增-微流控芯片电泳法同时检测多个SNP位点

以CYP2D6基因中的6个SNP位点为测定对象, 开展多个SNP位点同时测定的方法学研究.     

Highly multiplex and sensitive SNP genotyping method using a three‐color fluorescence‐labeled ligase detection reaction coupled with conformation‐sensitive CE

For the development of clinically useful genotyping methods for SNPs, accuracy, simplicity, sensitivity, and cost‐effectiveness are the most important criteria. Among the methods currently being developed for SNP genotyping technology, the ligation‐dependent method is considered the simplest for clinical diagnosis. However, sensitivity is not guaranteed by the ligation reaction alone, and analysis of multiple targets is limited by the detection method. Although CE is an attractive alternative to error‐prone hybridization‐based detection, the multiplex assay process is complicated because of the size‐based DNA separation principle. In this study, we employed the ligase detection reaction coupled with high‐resolution CE‐SSCP to develop an accurate, sensitive, and simple multiplex genotyping method. Ligase detection reaction could amplify ligated products through recurrence of denaturation and ligation reaction, and SSCP could separate these products according to each different structure conformation without size variation. Thus, simple and sensitive SNP analysis can be performed using this method involving the use of similar‐sized probes, without complex probe design steps. We found that this method could not only accurately discriminate base mismatches but also quantitatively detect 37 SNPs of the tp53 gene, which are used as targets in multiplex analysis, using three‐color fluorescence‐labeled probes.     

Stuffer‐free multiplex ligation‐dependent probe amplification based on conformation‐sensitive capillary electrophoresis: A novel technology for robust multiplex determination of copy number variation

Developing diagnostic tools based on the application of known disease/phenotype‐associated copy number variations (CNVs) requires the capacity to measure CNVs in a multiplex format with sufficient reliability and methodological simplicity. In this study, we developed a reliable and user‐friendly multiplex CNV detection method, termed stuffer‐free MLPA‐CE‐SSCP, that combines a variation of multiplex ligation‐dependent probe amplification (MLPA) with CE‐SSCP. In this variation, MLPA probes were designed without the conventionally required stuffer sequences. To separate the similar‐sized stuffer‐free MLPA products, we adopted CE‐SSCP rather than length‐dependent conventional CE analysis. An examination of the genomic DNA from five cell lines known to vary in X‐chromosome copy number (1–5) revealed that copy number determinations using stuffer‐free MLPA‐CE‐SSCP were more accurate than those of conventional MLPA, and the CV of the measured copy numbers was significantly lower. Applying our system to measure the CNVs on autosomes between two HapMap individuals, we found that all peaks for CNV targets showed the expected copy number changes. Taken together, our results indicate that this new strategy can overcome the limitations of conventional MLPA, which are mainly related to long probe length and difficulties of probe preparation.     

A multiplex single nucleotide polymorphism genotyping method using ligase‐based mismatch discrimination and CE‐SSCP

Accuracy, simplicity, and cost‐effectiveness are the most important criteria for a genotyping method for SNPs compatible with clinical use. One method developed for SNP genotyping, ligase‐based discrimination, is considered the simplest for clinical diagnosis. However, multiplex assays using this method are limited by the detection method. Although CE has been introduced as an alternative to error prone microarray‐based detection, the design process and multiplex assay procedure are complicated because of the DNA size‐dependent separation principle. In this study, we developed a simple and accurate multiplex genotyping method using reaction condition‐optimized ligation and high‐resolution CE‐based SSCP. With this high‐resolution CE‐SSCP system, we are able to use similar‐sized probes, thereby eliminating the complex probe design step and simplifying the optimization process. We found that this method could accurately discriminate single‐base mismatches in SNPs of the tp53 gene, used as targets for multiplex detection.     

A universal multiplex PCR strategy for 100-plex amplification using a hydrophobically patterned microarray

Both basic research and clinical medicine have urgent demands for highly efficient strategies to simultaneously identify many different DNA sequences within a single tube. Effective and simultaneous amplification of multiple target sequences is a prerequisite for any successful multiple nucleic acid detection method. Multiplex PCR is one of the best choices for this purpose. However, due to the intrinsic interference and competition among primer pairs in the same tube, multiple rounds of highly empirical optimization procedures are usually required to establish a successful multiplex PCR reaction. To address this challenge, we report here a universal multiplex PCR strategy that is capable of over 100-plex amplification using a specially designed microarray in which hydrophilic microwells are patterned on a hydrophobic chip. On such an array, primer pairs tagged with a universal sequence are physically separated in individual hydrophilic microwells on an otherwise hydrophobic chip, enabling many unique PCR reactions to be proceeded simultaneously during the first step of the procedure. The PCR products are then isolated and further amplified from the universal sequences, producing a sufficient amount of material for analysis by conventional gel electrophoresis or DNA microarray technology. This strategy is abbreviated as "MPH&HPM" for "Multiplex PCR on a Hydrophobically and Hydrophilically Patterned Microarray". The feasibility of this method is first demonstrated by a multiplex PCR reaction for the simultaneous detection of eleven pneumonia-causing pathogens. Further, we demonstrate the power of this strategy with a highly successful 116-plex PCR reaction that required only little prior optimization. The effectiveness of the MPH&HPM strategy with clinical samples is then illustrated with the detection of deleted exons of the Duchenne Muscular Dystrophy (DMD) gene, the results are in excellent agreement with the clinical records. Because of its generality, simplicity, flexibility, specificity and capacity of more than 100-plex amplification, the MPH&HPM strategy should have broad applications in both laboratory research and clinical applications when multiplex nucleic acid analysis is required.     

Determination of Genetically Modified Soybean by Multiplex PCR and CGE with LIF Detection

A method for rapid screening, identification and detection of genetically modified soybean by multiplex polymerase chain reaction (PCR) and capillary gel electrophoresis with laser-induced fluorescence (CGE–LIF) was developed and applied to actual food samples. A triplex PCR procedure was used to amplify the parts of nopaline synthase (NOS) terminator, and the junction between cauliflower mosaic virus 35S promoter and chloroplast transit peptide CTP4 trait gene, as well as the lectin gene to allow the screening and identification of specific transgenic soybean line (glyphosate-tolerant soybean). The multiplex PCR parameters and conditions of capillary gel electrophoresis were optimized. The amplified DNA fragments were analyzed by CGE–LIF. The amplified PCR products were analyzed by CGE–LIF within about 20 min. The method developed is highly sensitive and allows the detection of a percentage of genetically modified soybean as low as 0.025%. The percentage is low enough to fulfill the requirement of the EU Regulation for transgenic food labeling of 1.0%. The sequences of the multiple PCR products were identical with those published in Genbank. The proposed method has been used in identification and detection of genetically modified soybean in various food samples. Compared with agarose gel electrophoresis (AGE), the proposed method is more rapid, accurate and requires a smaller amount of samples. Thus an efficient alternative method is provided for monitoring genetically modified soybean in order to meet the increasing demand of implementation of the genetically modified food labeling policy.     

Quantitative analysis of DNA methylation in the promoter region of the methylguanine‐O6‐DNA‐methyltransferase gene by COBRA and subsequent native capillary gel electrophoresis

Along with histone modifications, RNA interference and delayed replication timing, DNA methylation belongs to the key processes in epigenetic regulation of gene expression. Therefore, reliable information about the methylation level of particular DNA fragments is of major interest. Herein the methylation level at two positions of the promoter region of the gene methylguanine‐O⁶‐DNA‐Methyltransferase (MGMT) was investigated. Previously, it was demonstrated that the epigenetic status of this DNA region correlates with response to alkylating anticancer agents. An automated CGE method with LIF detection was established to separate the six DNA fragments resulting from combined bisulfite restriction analysis of the methylated and non‐methylated MGMT promoter. In COBRA, the DNA was treated with bisulfite converting cytosine into uracil. During PCR uracil pairs with adenine, which changes the original recognition site of the restriction enzyme Taql. Artificial probes generated by mixing appropriate amounts of DNA after bisulfite treatment and PCR amplification were used for validation of the method. The methylation levels of these samples could be determined with high accuracy and precision. DNA samples prepared by mixing the corresponding clones first and then performing PCR amplification led to non‐linear correlation between the corrected peak areas and the methylation levels. This effect is explained by slightly different PCR amplification of DNA with different sequences present in the mixture. The superiority of CGE over PAGE was clearly demonstrated. Finally, the established method was used to analyze the methylation levels of human brain tumor tissue samples.     

Colorimetric quantification of mRNA expression in rare tumour cells amplified by multiple ligation-dependent probe amplification

An enzyme-linked oligonucleotide assay (ELONA) for quantification of mRNA expression of five genes involved in breast cancer, extracted from isolated rare tumour cells and amplified by multiplex ligation-dependent probe amplification (MLPA) is presented. In MLPA, a multiplex oligonucleotide ligation assay is combined with a PCR reaction in which all ligation products are amplified by use of a single primer pair. Biotinylated probes complementary to each of the target sequences were immobilised on the surface of a streptavidin-coated microtitre plate and exposed to single-stranded MLPA products. A universal reporting probe sequence modified with horseradish peroxidase (URP–HRP) and complementary to a universal primer used during the MLPA step was further added to the surface-bound duplex as a reporter probe. Simultaneous addition of anchoring probe and target, followed by addition of reporter probe, rather than sequential addition, was achieved with no significant effect on sensitivity and limits of detection, but considerably reduced the required assay time. Detection limits as low as 20 pmol L⁻¹, with an overall assay time of 95 min could be achieved with negligible cross-reactivity between probes and non-specific targets present in the MLPA-PCR product. The same MLPA-PCR product was analysed using capillary electrophoresis, the technique typically used for analysis of MLPA products, and good correlation was observed. The assay presented is easy to carry out, relatively inexpensive, rapid, does not require sophisticated instrumentation, and enables quantitative analysis, making it very promising for the analysis of MLPA products.     

Multiplex identification of drug‐resistant Gram‐positive pathogens using stuffer‐free MLPA system

Early detection of pathogens from blood and identification of their drug resistance are essential for sepsis management. However, conventional culture‐based methods require relatively longer time to identify drug‐resistant pathogens, which delays therapeutic decisions. For precise multiplex detection of drug‐resistant Gram‐positive pathogens, we developed a method by using stuffer‐free multiplex ligation‐dependent probe amplification (MLPA) coupled with high‐resolution CE single‐strand conformation polymorphisms (CE‐SSCP) system. We designed three probe sets for genes specific to Gram‐positive species (Staphylococcus aureus: nuc, Enterococcus faecium: sodA, and Streptococcus pneumoniae: lytA) and two sets for genes associated with drug resistance (mecA and vanA) to discriminate major Gram‐positive pathogens with the resistance. A total of 94 different strains (34 reference strains and 60 clinical isolates) were used to validate this method and strain‐specific peaks were successfully observed for all the strains. To improve sensitivity of the method, a target‐specific preamplification step was introduced and, consequently, the sensitivity increased from 10 pg to 100 fg. We also reduced a total assay time to 8 h by optimizing hybridization time without compromising test sensitivity. Taken together, our multiplex detection system can improve detection of drug‐resistant Gram‐positive pathogens from sepsis patients’ blood.     

A simple capillary gel electrophoresis approach for efficient and reproducible DNA separations. Analysis of genetically modified soy and maize

It is generally assumed that in order to achieve suitable separations of DNA fragments, capillary gel electrophoresis (CGE)-coated capillaries should be used. In this work, a new method is presented that allows to obtain reproducible CGE separations of DNA fragments using bare fused-silica capillaries without any previous coating step. The proposed method only requires: (i) a capillary washing with 0.1 M hydrochloric acid between injections and (ii) a running buffer composed of Tris-phosphate-ethylenediamine tetraacetic acid (EDTA) and 4.5% of 2-hydroxyethyl cellulose (HEC) as sieving polymer. The use of this new CGE procedure gives highly resolved and reproducible separations of DNA fragments ranging from 50 to 750 bp. The separation of these DNA fragments is accomplished in less than 30 min with efficiencies up to 1.7 x 10(6) plates/m. Reproducibility values of migration times (given as %RSD) for the analyzed DNA fragments are better than 1.0% (n = 4) for the same day, 2.2% (n = 16) for four different days, and 2.3% (n = 16) for four different capillaries. The usefulness of this separation method is demonstrated by detecting genetically modified maize and genetically modified soy after DNA amplification by PCR. This new CGE procedure together with LIF as detector provides sensitive analysis of 0.9% of Bt11 maize, Mon810 maize, and Roundup Ready soy in flours with S/ N up to 542. These results demonstrate the usefulness of this procedure to fulfill the European regulation on detection of genetically modified organisms in foods.     

Capillary and microelectrophoretic separations of ligase detection reaction products produced from low-abundant point mutations in genomic DNA

Capillary gel electrophoresis (CGE) and polymer-based microelectrophoretic platforms were investigated to analyze low-abundant point mutations in certain gene fragments with high diagnostic value for colorectal cancers. The electrophoretic separations were carried out on single-stranded DNA (ssDNA) products generated from an allele-specific ligation assay (ligase detection reaction, LDR), which was used to screen for a single base mutation at codon 12 in the K-ras oncogene. The presence of the mutation generated a ssDNA fragment that was >40 base pairs (bp) in length, while the primers used for the ligation assay were <30 bp in length. Various separation matrices were investigated, with the success of the matrix assessed by its ability to resolve the ligation product from the large molar excess of unligated primers when the mutant allele was lower in copy number compared to the wild-type allele. Using CGE, LDR product models (44 and 51 bp) could be analyzed in a cross-linked polyacrylamide gel with a 1000-fold molar excess of LDR primers (25 bp) in approximately 45 min. However, when using linear polyacrylamide gels, these same fragments could not be detected due to significant electrokinetic biasing during injection. A poly(methylmethacrylate) (PMMA) microchip of 3.5 cm effective column length was used with a 4% linear polyacrylamide gel to analyze the products generated from an LDR. When the reaction contained a 100-fold molar excess of wild-type DNA compared to a G12.2D mutant allele, the 44 bp ligation product could be effectively resolved from unligated primers in under 120 s, nearly 17 times faster than the CGE format. In addition, sample cleanup was simplified using the microchip format by not requiring desalting of the LDR prior to loading.