Approaching real-time molecular diagnostics: single-pair fluorescence resonance energy transfer (spFRET) detection for the analysis of low abundant point mutations in K-ras oncogenes

The aim of this study was to develop new strategies for analyzing molecular signatures of disease states approaching real-time using single pair fluorescence resonance energy transfer (spFRET) to rapidly detect point mutations in unamplified genomic DNA. In addition, the detection process was required to discriminate between normal and mutant (minority) DNAs in heterogeneous populations. The discrimination was carried out using allele-specific primers, which flanked the point mutation in the target gene and were ligated using a thermostable ligase enzyme only when the genomic DNA carried this mutation. The allele-specific primers also carried complementary stem structures with end-labels (donor/acceptor fluorescent dyes, Cy5/Cy5.5, respectively), which formed a molecular beacon following ligation. We coupled ligase detection reaction (LDR) with spFRET to identify a single base mutation in codon 12 of a K-ras oncogene that has high diagnostic value for colorectal cancers. A simple diode laser-based fluorescence system capable of interrogating single fluorescent molecules undergoing FRET was used to detect photon bursts generated from the molecular beacon probes formed upon ligation. LDR-spFRET provided the necessary specificity and sensitivity to detect single-point mutations in as little as 600 copies of human genomic DNA directly without PCR at a level of 1 mutant per 1000 wild type sequences using 20 LDR thermal cycles. We also demonstrate the ability to rapidly discriminate single base differences in the K-ras gene in less than 5 min at a frequency of 1 mutant DNA per 10 normals using only a single LDR thermal cycle of genomic DNA (600 copies). Real-time LDR-spFRET detection of point mutations in the K-ras gene was accomplished in PMMA microfluidic devices using sheath flows.     

A novel one cycle allele specific primer extension--molecular beacon displacement method for DNA point mutation detection with improved specificity

We report here a new method for the real-time detection of DNA point mutations with molecular beacon as the fluorescence tracer and 3′ (exo-) Bst DNA polymerase large fragment as the polymerase. The method is based on the mechanism of allele specific primer extension-strand displacement (ASPE-SD). To improve the specificity of the method only one cycle of the allele specific polymerase chain reaction (PCR) was used that could largely eliminate the non-specific reactions between the primers and template of the “wrong” genotype. At first, the primer and molecular beacon both hybridize to the DNA template, and the molecular beacon emits intensive fluorescence. The role of 3′ exonuclease excision of Bst DNA polymerase large fragment is utilized for primer extension. When 3′-termini matches its corresponding template, the primer would efficiently extend and replace the molecular beacon that would simultaneously return to its closed form leading to the quenching of the fluorescence. However, when 3′-termini of the primer mismatches its corresponding template primer extension and molecular beacon displacement would not happen and fluorescence of the hybridized molecular beacon holds the line without fluorescence quenching. This approach was fully demonstrated in synthetic template systems and applied to detect point mutation at codon 259, a possible point mutation site in exon 7 of p53 gene, obtained from human genomic DNA samples with unambiguous differentiation power.     

微流控芯片电泳-多重聚合酶链反应法同时测定多个单碱基多态性位点

以微流控芯片电泳为检测平台,建立了多重PCR扩增法同时测定多个单碱基多态性(SNP)位点的方法。先通过PCR扩增得一段含所有待测SNP位点的长片段;用限制性内切酶消化成短片段,再将酶切反应产物与脱氧核糖核酸适配器(DNAadapter)相连;以连接产物为模板,分成两管,分别用n条等位基因特异性引物和一条通用引物进行n重PCR扩增;最后用微流控芯片电泳法分离PCR扩增产物,根据两管扩增产物的芯片电泳图谱中扩增片段的大小判断SNP的类型。以细胞色素P4502D6(CYP2D6)基因中的5个SNP位点(100C>T、1661G>C、1758G>T、2470T>C和2850C>T)为检测对象,考察了各等位基因特异性引物之间的相互影响和扩增反应的特异性,采用微流控芯片电泳法成功测定了20名健康中国人的CYP2D6基因中5个SNP位点的基因多态性,与聚合酶链反应-限制性片段长度多态性法(PCR-RFLP)测定结果完全一致。     

RNA-templated single-base mutation detection based on T4 DNA ligase and reverse molecular beacon

A novel RNA-templated single-base mutation detection method based on T4 DNA ligase and reverse molecular beacon (rMB) has been developed and successfully applied to identification of single-base mutation in codon 273 of the p53 gene. The discrimination was carried out using allele-specific primers, which flanked the variable position in the target RNA and was ligated using T4 DNA ligase only when the primers perfectly matched the RNA template. The allele-specific primers also carried complementary stem structures with end-labels (fluorophore TAMRA, quencher DABCYL), which formed a molecular beacon after RNase H digestion. One-base mismatch can be discriminated by analyzing the change of fluorescence intensity before and after RNase H digestion. This method has several advantages for practical applications, such as direct discrimination of single-base mismatch of the RNA extracted from cell; no requirement of PCR amplification; performance of homogeneous detection; and easily design of detection probes.     

A novel technique for detecting single nucleotide polymorphisms by analyzing consumed allele-specific primers

We present a simple and rapid polymerase chain reaction (PCR)-based technique, termed consumed allele-specific primer analysis (CASPA), as a new strategy for single nucleotide polymorphism (SNP) analysis. The method involves the use of labeled allele-specific primers, differing in length, with several noncomplementary nucleotides added in the 5'-terminal region. After PCR amplification, the amounts of the remaining primers not incorporated into the PCR products are determined. Thus, nucleotide substitutions are identified by measuring the consumption of primers. In this study, the CASPA method was successfully applied to ABO genotyping. In the present method, the allele-specific primer only anneals with the target polymorphic site on the DNA, so it is not necessary to analyze the PCR products. Therefore, this method is only little affected by modification of the PCR products. The CASPA method is expected to be a useful tool for typing of SNPs.     

Design of allele-specific primers and detection of the human ABO genotyping to avoid the pseudopositive problem

PCR experiments using DNA primers forming mismatch pairing with template lambda DNA at the 3' end were carried out in order to develop allele-specific primers capable of detecting SNP in genomes without generating pseudopositive amplification products, and thus avoiding the so-called pseudopositive problem. Detectable amounts of PCR products were obtained when primers forming a single or two mismatch pairings at the 3' end were used. In particular, 3' terminal A/C or T/C (primer/template) mismatches tended to allow PCR amplification to proceed, resulting in pseudopositive results in many cases. While less PCR product was observed for primers forming three terminal mismatch pairings, target DNA sequences were efficiently amplified by primers forming two mismatch pairings next to the terminal G/C base pairing. These results indicate that selecting a primer having a 3' terminal nucleotide that recognizes the SNP nucleotide and the next two nucleotides that form mismatch pairings with the template sequence can be used as an allele-specific primer that eliminates the pseudopositive problem. Trials with the human ABO genes demonstrated that this primer design is also useful for detecting a single base pair difference in gene sequences with a signal-to-noise ratio of at least 45.     

Microarray-based resequencing by apyrase-mediated allele-specific extension

We have developed an array-based resequencing method to determine genetic alterations in putative cancer genes. The method relies on that the specificity of DNA polymerase in allele-specific extensions can be enhanced by terminating the extension reactions with apyrase and that a tiling set of primers are synthesized covering the investigated gene sequence. We report on such apyrase-mediated allele-specific primer extension (AMASE) assay as a method suitable for high-throughout resequencing and mutation detection in tumor suppressor genes and oncogenes. In the experimental setup, primers complementary to codons 12, 13 and codon 61 of the N-ras proto-oncogene were spotted onto glass slides. Overlapping sense and anti-sense primers were designed so that complementary primers for all possible mutations in each base position were investigated. The extension reactions were performed in a single step following hybridization of target DNA to the immobilized primers on the array surface. Mutation detection limits and the possibility of quantifying the mutations were investigated using synthetic oligonucleotides. In addition, 64 clinical samples were sequenced and 16 of these showed mutations in the N-ras gene.     

未修饰的纳米金结合等位特异性扩增来检测K-ras基因点突变

将等位基因特异性扩增的特异性与纳米金特殊的光学性质相结合, 发展了一种新的基因点突变检测方法. 以肿瘤中常见的K-ras癌基因第12位密码子作为点突变检测对象, 采用突变型引物对待测序列进行等位特异性扩增. 突变型样品扩增产物中大部分是双链DNA; 而野生型样品由于不能被顺利扩增, 产物中大部分是单链DNA. 以纳米金颗粒作为报告基团, 向两种不同基因型扩增产物中依次加入纳米金胶和盐溶液, 野生型基因扩增产物中的单链引物被吸附到纳米金颗粒表面, 使得纳米金在适宜浓度的盐溶液中不发生聚集; 突变型样品扩增产物中的双链DNA由于与纳米金颗粒间存在静电斥力而不能被吸附到纳米金颗粒表面, 纳米金在该浓度的盐溶液中发生聚集, 导致两种基因型的混合液在吸收光谱和颜色方面均存在显著差异, 从而实现了检测基因点突变的目的. 该检测方法直观、快速、简便, 实验成本低, 能够检测到pmol量级的样品, 为点突变检测提供了一种实用的新方法.     

Development of a ligase detection reaction/CGE method using a LIF dual‐channel detection system for direct identification of allelic composition of mutated DNA in a mixed population of excess wild‐type DNA

We developed an inexpensive LIF dual‐channel detection system and applied it to a ligase detection reaction (LDR)/CGE method to identify the allelic composition of low‐abundance point mutations in a large excess of wild‐type DNA in a single reaction with a high degree of certainty. Ligation was performed in a tube with a nonlabeled common primer and multiplex discriminating primers, each labeled with a different standard fluorophore. The discriminating primers were directed against three mutant variations in codon 12 of the K‐ras oncogene that have a high diagnostic value for colorectal cancer. LDR products generated from a particular K‐ras mutation through successful ligation events were separated from remaining discriminating primers by CGE, followed by LIF detection using the new system, which consists of two photomultiplier tubes, each with a unique optical filter. Each fluorophore label conjugated to the corresponding LDR product produced a distinct fluorescence signal intensity ratio from the two detection channels, allowing spectral discrimination of the three labels. The ability of this system to detect point mutations in a wild‐type sequence‐dominated population, and to disclose their allelic composition, was thus demonstrated successfully.     

Serial processing of biological reactions using flow-through microfluidic devices: coupled PCR/LDR for the detection of low-abundant DNA point mutations

We have fabricated a flow-through biochip consisting of passive elements for the analysis of single base mutations in genomic DNA using polycarbonate (PC) as the substrate. The biochip was configured to carry out two processing steps on the input sample, a primary polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) for scoring the presence of low abundant point mutations in genomic DNA. The operation of the device was demonstrated by detecting single nucleotide polymorphisms in gene fragments (K-ras) that carry high diagnostic value for colorectal cancers. The effect of carryover from the primary PCR on the subsequent LDR was investigated in terms of LDR yield and fidelity. We found that a post-PCR treatment step prior to the LDR phase of the assay was not essential. As a consequence, a thermal cycling microchip was used for a sequential PCR/LDR in a simple continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of the gene fragments from genomic DNA; (2) mixing of the resultant PCR product(s) with an LDR cocktail via a Y-shaped passive micromixer; and (3) ligation of two primers (discriminating primer that carried the complement base to the mutation locus being interrogated and a common primer) only when the particular mutation was present in the genomic DNA. We successfully demonstrated the ability to detect one mutant DNA in 1000 normal sequences with the integrated microfluidic system. The PCR/LDR assay using the microchip performed the entire assay at a relatively fast processing speed: 18.7 min for 30 rounds of PCR, 4.1 min for 13 rounds of LDR (total processing time = ca. 22.8 min) and could screen multiple mutations simultaneously in a multiplexed format. In addition, the low cost of the biochip due to the fact that it was fabricated from polymers using replication technologies and consisted of passive elements makes the platform amenable to clinical diagnostics, where one-time use devices are required to eliminate false positives resulting from carryover contamination.     

Extension of the GOOD assay for genotyping single nucleotide polymorphisms by matrix-assisted laser desorption/ionization mass spectrometry

Over the past years several methods using mass spectrometry for high-throughput genotyping of single nucleotide polymorphisms (SNPs) have been developed. Most of these procedures require stringent purification. Only the GOOD assay does not need any sample purification. Here, several new implementations of this assay are presented. The molecular biological procedure of the GOOD assays is based on the principle that the analysis of DNA by matrix-assisted laser desorption/ionization (MALDI) is strongly dependent on the charge state. A 100-fold increase in sensitivity can be achieved if the analyzed DNA product is conditioned by a chemical procedure termed 'charge-tagging'. The GOOD assay starts with a PCR; allele-specific DNA molecules are generated by extension of modified primers. These contain up to three phosphorothioates and optionally a quaternary ammonium charged group with ddNTPs or alpha-S-ddNTPs. Then the unmodified part of the primers is digested by phosphodiesterase II and the negative charges of the phosphorothioates are neutralized by an alkylation reaction resulting in charge-tagged DNA products. Through the use of a novel DNA polymerase for the primer extension, which preferably incorporates ddNTPs over dNTPs, an enzymatic degradation of residual dNTPs from the PCR is not required. Additionally, the unique property of charge-tag technology is demonstrated to detect specifically on the same sample allele-specific DNA products carrying a positive charge-tag in the positive ion mode while products carrying a negative charge-tag are analyzed in the negative ion mode. We also generated zwitterionic allele-specific products that were detectable with high sensitivity in positive ion mode. The findings of this study raise interesting questions about the ionization process of nucleic acids in MALDI. The new variations of the GOOD assay were applied to genotype SNPs of a candidate gene for cardiovascular disease.     

Microarray-based detection of Korean-specific <Emphasis Type="Italic">BRCA1</Emphasis> 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. Cheulhee Jung and Seong-Chun Yim contributed equally to this work.     

Multiple sample amplification and genotyping integrated on a single electronic microarray

We report a novel method that allows simultaneous in situ amplification and then genotyping of single nucleotide polymorphism (SNP) for multiple samples on a single electronic microarray. The locus coding for one of the common inherited thrombosis risk factors, Factor V Leiden (FVL), was chosen as a model system for SNP analysis. This method combines strand displacement amplification (SDA) with electrophoretic movement and concentration of DNA on electronic microarrays to provide a single platform for DNA amplification and analysis. The method includes: electronic anchoring of allele-specific SDA amplifiable primers (APs) and a nonamplifiable primer (NAP) to different electrodes, electronic hybridization of genomic DNA from different samples to those primers, in situ amplification of target DNA, and genotyping of FVL. Compared to previous anchored SDA methods, the addition of a NAP improves detection signals by at least 20-fold. The sensitivity of this method is dependent on the amplification time. Using this method, nine different genomic DNA samples with known FVL genotypes were amplified and correctly genotyped on a single electronic microarray without any contamination between samples. The present method could streamline development of nucleic acid-based assays in applications of molecular diagnostic, point-of-care testing, and forensic detection, which often require the capability to analyze multiple samples efficiently.     

Genotyping of K-ras codons 12 and 13 mutations in colorectal cancer by capillary electrophoresis

Point mutations of the K-ras gene located in codons 12 and 13 cause poor responses to the anti-epidermal growth factor receptor (anti-EGFR) therapy of colorectal cancer (CRC) patients. Besides, mutations of K-ras gene have also been proven to play an important role in human tumor progression. We established a simple and effective capillary electrophoresis (CE) method for simultaneous point mutation detection in codons 12 and 13 of K-ras gene. We combined one universal fluorescence-based nonhuman-sequence primer and two fragment-oriented primers in one tube, and performed this two-in-one polymerase chain reaction (PCR). PCR fragments included wild type and seven point mutations at codons 12 and 13 of K-ras gene. The amplicons were analyzed by single-strand conformation polymorphism (SSCP)-CE method. The CE analysis was performed by using a 1× Tris–borate–EDTA (TBE) buffer containing 1.5% (w/v) hydroxyethylcellulose (HEC) (MW 250 000) under reverse polarity with 15 °C and 30 °C. Ninety colorectal cancer patients were blindly genotyped using this developed method. The results showed good agreement with those of DNA sequencing method. The SSCP-CE was feasible for mutation screening of K-ras gene in populations.     

Sensitive and enzyme-free detection for single nucleotide polymorphism using microbead-assisted toehold-mediated strand displacement reaction

This report described a free-enzyme, convenient and inexpensive genotyping biosensor capable of detecting single nucleotide polymorphism at normal temperature based on the combination of toeholdmediated strand displacement reaction(toehold-SDR) and microbead-capture technique. The biosensor consists of a pre-hybridized strand formed by a reporter probe and a capture probe. In the presence of a mutant sequence, there is no toehold-mediated strand displacement and the reporter probe cannot be released from the pre-hybridized strand. Microbeads capture the fluorescent pre-hybridized strand through biotin–streptavidin interaction, so microbeads give out significant fluorescence signal, while there is no fluorescence in the solution. However, in the presence of a matched target, the strand displacement is effectively initiated and the reporter probe is released from pre-hybridized strand. After adding microbeads, the solution produces bright fluorescence, while microbeads have no obvious signal.Genotypes are identified conveniently according to the fluorescence intensity of the solution. The method provides a simple and inexpensive strategy to detect point mutation. Moreover, this biosensor shows the linear relationship in the range of 1–40 nmol/L and reaches a detection limit of 0.3 nmol/L.     

电化学发光-聚合酶链式反应方法检测胰腺癌中K-ras基因点突变

发展了一种可用于快速检测胰腺癌中K-ras癌基因点突变的电化学发光-聚合酶链式反应(ECL-PCR)分析方法。该法采用三联吡啶钌标记的上游引物和生物素标记的下游引物对目的片段进行PCR扩增;再采用限制性内切酶MvaI对扩增产物进行酶切。由于野生型样品和突变型样品间存在酶切位点的变化,其中只有野生型样品能被切断;通过生物素与链霉亲和素包被的磁珠连接,将生物素标记的DNA片段收集到检测池中,进行电化学发光检测。采用该法对13例胰腺癌组织中的K-ras癌基因第12位密码子进行点突变分析,只需要10μL样品、20min孵育时间和30s采集时间,就可得出其中有12例存在点突变,点突变率为92.3%。本方法操作简便、安全、快速、灵敏,可用于检测任何一种导致限制性内切酶位点改变的基因点突变。     

Allele-specific extension on microarray for DNA methylation analysis

Aberrant DNA methylation of CpG site in the gene promoter region has been confirmed to be closely associated with carcinogenesis. In this present study, a new method based on the allele-specific extension on microarray technique for detecting changes of DNA methylation in cancer was developed. The target gene regions were amplified from the bisulfite treated genomic DNA (gDNA) with modified primers and treated with exonuclease to generate single-strand targets. Allele-specific extension of the immobilized primers took place along a stretch of target sequence with the presence of DNA polymerase and Cy5-labeled dGTP. To control the false positive signals, the hybridization condition, DNA polymerase, extension time and primers design were optimized. Two breast tumor-related genes (P16 and E-cadherin) were analyzed with this present method successfully and all the results were compatible with that of traditional methylation-specific PCR. The experiments results demonstrated that this DNA microarray-based method could be applied as a high throughput tool for methylation status analysis of the cancer-related genes, which could be widely used in cancer diagnosis or the detection of recurrence.     

Improved adapter-ligation-mediated allele-specific amplification for multiplex genotyping by using software

Adapter-ligation-mediated allele-specific amplification (ALM-ASA) is a potential method for multiplex SNPs typing at an ultra low cost. Here, we describe a kind of software, which designs allele-specific primers for ALM-ASA assay on multiplex SNPs. DNA sequences containing SNPs of interest are submitted into the software which contains various endonucleases for options. Based on the SNP sequence information and the selected endonucleases, the software is capable of automatically generating sets of information needed to perform genotyping experiments. Each set contains a suitable endonuclease, qualified allele-specific primers with orientations and melting temperatures, sizes of allele-specific amplicons, and gel electropherograms simulated according to the sizes of the allele-specific amplicons and the mobility of DNA fragments in 2% agarose gel. Seven SNPs in the arylamines N-acetyltransferase 2 (NAT2) gene, five SNPs in the BRCA1 gene, five SNPs in the COMT gene, six SNPs in the CYP2E1 gene, five SNPs in the MPO gene, and six SNPs in the NRG1 gene were selected for evaluating the software. Without extra optimization, seven SNPs in the NAT2 gene were successfully genotyped for genomic DNA samples from 127 individuals by using the first set of allele-specific primers yielded by the software. Although several steps are used in the ALM-ASA assay, the whole genotyping process can be completed within 3 h by optimizing each step. Profiting from the software, the ALM-ASA assay is easy-to-perform, labor-saving, and accurate.     

Discrimination of A1555G and C1494T point mutations in the mitochondrial 12S rRNA gene by on/off switch

The objective of this study was to apply the “on/off” switch consisting of 3′ phosphorothioate-modified allele specific primers and exo⁺ polymerase in single base discrimination of A1555G and C1494T mutations in the highly conserved sites of the mitochondrial 12S rRNA. The two point mutations are the hotspot mutations associated with either aminoglycoside antibiotics induced deafness or inherited nonsyndromic hearing loss. The PCR products of mitochondrial DNA (mtDNA) 12S rRNA gene were inserted into the pMD19-T vector for transformation into Escherichia coli JM109 competent cells for preparing wild-type pMD19-T/mt vector. Inverse PCR was carried out for mtDNA 12S rRNA gene C1494T and A1555G mutagenesis and DpnI endonuclease degradating methylated pMD19-T/mt vector existing in the inverse PCR products was carried out to construct the mutation-type pMD19-T/mtM vector. These constructed vectors were confirmed by DNA sequencing. Allelic specific primers targeting wild-type and mutation-type templates were designed with 3′ terminal phosphorothioate modification. Two-directional primer extension was performed using Pfu polymerases. Amplified by exo⁺ polymerase, allelic specific primers perfectly matching wild-type allele were extended while no products were produced from primers targeting point-mutated deafness-related allele. Similarly, allelic specific primers perfectly matching point-mutated deafness-related mutation-type allele were extended and no products were yielded from primers targeting wild-type allele. No specific product was observed in the primer extension reaction mediated by on/off switch in screening the mtDNA 12S rRNA gene harboring either C1494T or A1555G mutation in 40 healthy volunteers tested. These data suggest that the “off switch” mediated by exo⁺ polymerase is highly reliable in the diagnosis of monogenic diseases and the novel “on/off” switch has enormous applications in systematic and extended screening of the12S rRNA gene A1555G and C1494T mutations. The established assay can be widely used not only for hearing loss patients but also for normal subjects before the use of aminoglycoside antibiotics.     

Lateral flow dipstick test for genotyping of 15 beta-globin gene (HBB) mutations with naked-eye detection

For definitive diagnosis of thalassemia carriers and patients, as well as for prenatal diagnosis, genotype analysis is of fundamental importance. We report a dry-reagent, lateral flow dipstick test that enables visual genotyping (detection by naked eye) of 15 mutations common in Mediterranean populations in the beta-globin gene (HBB). The method comprises 3 simple steps: (i) PCR amplification of a single 1896 bp segment of the beta globin gene flanking all 15 mutations; (ii) a multiplex (10-plex and/or 30-plex) primer extension reaction of the unpurified amplification product using allele-specific primers. Biotin is incorporated in the extended product; (iii) a dry-reagent multi-allele (10-plex) dipstick assay for visual detection of the primer extension reaction products within minutes. The total time required for PCR, primer extension reaction and the dipstick assay is ∼2 h. The method was evaluated by genotyping 45 DNA samples of known genotypes and 54 blind samples. The results were fully concordant with reference methods. The method is simple, rapid, and cost-effective. Detection by the dipstick assay does not require specialized instrumentation or highly qualified personnel. The proposed method could be a particularly useful tool in laboratories with limited resources and a basis for point-of-care diagnostics especially in combination with PCR amplification from whole blood.