Semiautomated DNA mutation analysis using a robotic workstation and molecular beacons

BACKGROUND: Our increasing knowledge of the genetic basis of inheritable diseases requires the development of automated reliable methods for high-throughput analyses. METHODS: We investigated the combination of semiautomated DNA extraction from blood using a robotic workstation, followed by automated mutation detection using highly specific fluorescent DNA probes, so-called molecular beacons, which can discriminate between alleles with as little as one single-base mutation. We designed two molecular beacons, one recognizing the wild-type allele and the other the mutant allele, to determine genotypes in a single reaction. To evaluate this procedure, we examined the C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene, which is associated with an increased risk for cardiovascular disease and neural tube defects. DNA was isolated from 10 microL of fresh EDTA-blood samples by use of a robotic workstation. The DNA samples were analyzed using molecular beacons as well as conventional methods. RESULTS: Both methods were compared, and no differences were found between outcomes of genotyping. CONCLUSIONS: The described assay enables robust and automated extraction of DNA and analysis of up to 96 samples (10 microL of blood per sample) within 5 h. This is superior to conventional methods and makes it suitable for high-throughput analyses.     

Hepatitis B virus precore mutation and fulminant hepatitis in the United States. A polymerase chain reaction-based assay for the detection of specific mutation.

Hepatitis B virus (HBV) variants with precore mutation(s) resulting in the absence of HBeAg production have been associated with the occurrence of fulminant hepatitis in Japan, Israel, and southern Europe, where the prevalence of this HBV strain appears common. In areas such as United States, where HBV infection is not endemic, the role of this mutant virus in fulminant hepatitis is unknown. We developed an amplification refractory mutation detection system to detect specifically the presence of the G to A mutation at nucleotide position 1898, which is the most frequently observed mutation resulting in a precore stop codon. In addition, this method provided a quantitative measurement of the relative ratio of one strain to the other. Using this system, we tested HBV strains for the presence of the stop codon mutation in sera from 40 cases of fulminant hepatitis B occurring in the United States. Serum HBV DNAs from 28 patients were analyzed successfully. A mixture of wild-type and mutant strains in various ratios were observed in 15 patients, wild type exclusively in 11, and mutant exclusively in 2. Four of these patients had undergone liver transplantation for HBV-associated cirrhosis and developed fulminant HBV-associated hepatitis after transplantation. Pre- and posttransplant serum samples from one patient were analyzed: a mixture of wild-type and mutant HBV strains was detected in both samples. Our study demonstrated that both wild-type and mutant HBV strains are associated with fulminant hepatitis, and that in some patients in the United States, factors other than precore mutations contribute to the development of fulminant hepatitis.     

Detection and quantification of heteroplasmic mutant mitochondrial DNA by real-time amplification refractory mutation system quantitative PCR analysis: a single-step approach

BACKGROUND: The A3243G mitochondrial tRNA leu(UUR) point mutation causes mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome, the most common mitochondrial DNA (mtDNA) disorder, and is also found in patients with maternally inherited diabetes and deafness syndrome (MIDD). To correlate disease manifestation with mutation loads, it is necessary to measure the percentage of the A3243G mtDNA mutation. METHODS: To reliably quantify low proportions of the mutant mtDNA, we developed a real-time amplification refractory mutation system quantitative PCR (ARMS-qPCR) assay. We validated the method with experimental samples containing known proportions of mutant A3243G mtDNA generated by mixing known amounts of cloned plasmid DNA containing either the wild-type or the mutant sequences. RESULTS: A correlation coefficient of 0.9995 between the expected and observed values for the proportions of mutant A3243G in the experimental samples was found. Evaluation of a total of 36 patient DNA samples demonstrated consistent results between PCR-restriction fragment length polymorphism (RFLP) analysis and real-time ARMS-qPCR. However, the latter method was much more sensitive for detecting low percentages of mutant heteroplasmy. Three samples contained allele-specific oligonucleotide-detectable but RFLP-undetectable mutations. CONCLUSIONS: The real-time ARMS-qPCR method provides rapid, reliable, one-step quantitative detection of heteroplasmic mutant mtDNA.     

Rapid detection of the hepatitis B virus YMDD mutant using TaqMan-minor groove binder probes

BACKGROUND: TaqMan-minor groove binder (MGB) probes were used in a real-time PCR-based assay for the rapid and accurate detection of hepatitis B virus (HBV) YMDD mutants. METHODS: TaqMan-MGB probes were designed to distinguish between wild-type (YMDD) and mutant (YVDD and YIDD) strains of HBV. The detection limit and sensitivity of the assay were determined using a dilution series of a mixture of wild-type and mutant plasmids. Serum samples collected from four patients with chronic mutant HBV infections during lamivudine therapy were analyzed using this method. RESULTS: The detection limit for YVDD and YIDD was 10 and 50 copies, respectively, whereas the sensitivity was 10% within a mixed virus population. In the clinical samples, mutant strains of HBV could be detected at levels <2.6 log copies/ml of HBV DNA. While 15 of the 21 samples tested by this method were positive for the YMDD mutant, direct sequencing and a reverse hybridization line probe assay (INNO-LiPA HBV DR v2) detected the mutant strain in only 11 and 9 samples, respectively. Moreover, the data for 6 samples analyzed by TA cloning were fully consistent with our TaqMan PCR results. CONCLUSIONS: We successfully established a sensitive and accurate assay for the YMDD mutant of HBV. This method may be useful for monitoring patients treated with lamivudine.     

K-ras point mutation detection in lung cancer: comparison of two approaches to somatic mutation detection using ARMS allele-specific amplification

BACKGROUND: The use of sensitive molecular techniques to detect rare cells in a population is of increasing interest to the molecular pathologist, but detection limits often are poorly defined in any given molecular assay. We combined the approaches of real-time quantitative PCR with ARMS(TM) allele-specific amplification in a novel assay for detecting mutant K-ras sequences in clinical samples. METHODS: ARMS reactions were used to detect seven commonly occurring mutations in the K-ras oncogene. These mutations produce amino acid changes in codon 12 (Gly to Ala, Arg, Asp, Cys, Ser, or Val) and codon 13 (Gly to Asp). A control reaction was used to measure the total amount of amplifiable K-ras sequence in a sample so that the ratio of mutant to wild-type sequence could be measured. Quantitative data were confirmed for a selection of samples by an independent cloning and sequencing method. The assay was used to analyze 82 lung tumor DNA samples. RESULTS: The assay detected K-ras mutations in 44% of adenocarcinomas, which is equivalent to frequencies reported in the literature using ultrasensitive techniques. Forty-six percent of squamous carcinomas were also positive. The ratio of mutant sequence in the tumor DNA samples was 0.04-100%. CONCLUSIONS: The assay is homogeneous, with addition of tumor DNA sample being the only step before results are generated. The quantitative nature of the assay can potentially be used to define the analytical sensitivity necessary for any specified diagnostic application of K-ras (or other) point mutation detection.     

探针熔解分析法检测结核分枝杆菌异烟肼耐药突变的应用和评价

目的 评价PMA技术检测MTB对INH耐药突变的价值,调查INH耐药突变发生特征.方法 MTB标准株H37Rv来自国家结核病参比实验室,1株INH敏感株和1株katG S315TACC突变株来自厦门市疾病预防控制中心,7株含有已知INH耐药突变的结核耐药株来自深圳慢性病防治中心、河南省疾病预防控制中心、中国人民解放军第309医院和厦门市疾病预防控制中心.707份MTB临床分离株来自厦门市疾病预防控制中心、厦门市第一医院和漳州市疾病预防控制中心,126份MTB涂阳痰标本来自厦门市同安区疾病预防控制中心.MTB标准株H37Rv、7株MTB INH耐药株和833份临床标本均采用厦门致善结核分枝杆菌异烟肼耐药突变检测试剂盒热裂解法提取基因组DNA,1株INH敏感株和1株katG S315T ACC突变株采用AxyPrepTM细菌基因组DNA提取试剂盒提取基因组DNA.熔解曲线分析所检标本与野生型对照在katG315位密码子、inhA启动子区(- 17～-8位点)、ahpC启动子区(-44～-30以及-15 ～3位点)及inhA94位密码子的熔解温度(Tm值)差异判断标本是否发生INH耐药突变.3×105拷贝/反应的野生株和katG S315T ACC突变株以10倍梯度稀释至300拷贝/反应,分析PMA技术的灵敏度.用PMA技术检测7株含有已知INH耐药突变的结核耐药株,评价特异性,并就其中5种耐药突变进行重复性检验.测序验证PMA技术对833份标本INH耐药性的临床检测效能.结果 PMA技术从核酸提取到结果判断可在3小时内完成,在标准96孔实时PCR仪器上可同时检测46份标本.对野生株和katG S315T ACC突变株的灵敏度均为300拷贝/反应,能够同时区分9种INH耐药相关点突变或缺失,5种耐药突变的Tm值标准偏差均在0.5℃之内.检出的162份突变标本与测序验证结果均一致.临床标本验证突变率为19.4％( 162/833),在所检出的14种INH耐药突变katGS315T( AGC →ACC)、inhA启动子区- 15C→T和katG S315N (AGC→AAC)这3种突变占INH耐药突变标本的83.3％(135/162).结论 PMA技术可快速、灵敏、特异检测结核INH耐药突变.     

Linked linear amplification for simultaneous analysis of the two most common hemochromatosis mutations

BACKGROUND: Two mutations in HFE, G845A (amino acid substitution C282Y) and C187G (H63D), are associated with hereditary hemochromatosis. We developed and validated a novel method, linked linear amplification (LLA), for detection of these two mutations. METHODS: Two segments of HFE were amplified by a multiplex LLA reaction that generated biotinylated LLA products. Aliquots of the multiplex LLA reaction were captured in microwells by hybridization to immobilized allele-specific oligonucleotides (ASOs). One wild-type and one mutant ASO represented the DNA sequence at each of the two mutation sites. Hybridization was detected by a streptavidin-horseradish peroxidase-based colorimetric method. Genotypes obtained by LLA and PCR-restriction fragment length polymorphism (PCR-RFLP) methods for 320 individuals were compared. RESULTS: The amplified samples included the following genotypes as determined by PCR-RFLP: wild-type 282 and 63 codons (n = 105), C282Y homozygous mutant (n = 54), C282Y heterozygous (n = 52), H63D homozygous mutant (n = 17), H63D heterozygous (n = 59), and compound H63D and C282Y heterozygous mutant (n = 33). There was complete concordance between the results obtained by LLA and those obtained by PCR-RFLP analysis. The presence of another HFE mutation, A193T (encoding S65C), did not interfere with genotyping at codon 63. CONCLUSIONS: LLA provides a reliable method to detect the common mutations in HFE that cause hereditary hemochromatosis.     

Detection of common disease-causing mutations in mitochondrial DNA (mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes MTTL1 3243 A>G and myoclonic epilepsy associated with ragged-red fibers MTTK 8344A>G) by real-time polymerase chain reaction

The 3243A>G mutation in the MTTL1 (tRNA(Leu)) gene and the 8344A>G mutation in the MTTK (tRNA(Lys)) gene are the most common mutations found in mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes and myoclonic epilepsy associated with ragged-red fibers, respectively. These mitochondrial DNA mutations are usually detected by conventional polymerase chain reaction followed by restriction enzyme digestion and gel electrophoresis. We developed a LightCycler real-time polymerase chain reaction assay to detect these two mutations based on fluorescence resonance energy transfer technology and melting curve analysis. Primers and fluorescence-labeled hybridization probes were designed so that the sensor probe spans the mutation site. The observed melting temperatures differed in the mutant and wild-type DNA by 9 degrees C for the MTTL1 gene and 6 degrees C for the MTTK gene. This method correctly identified all 10 samples that were 3243A>G mutation-positive, all 4 samples that were 8344A>G mutation-positive, and all 30 samples that were negative for both mutations, as previously identified by traditional gel-based methods. This LightCycler assay is a rapid and reliable technique for molecular diagnosis of these mitochondrial gene mutations.     

DGGE analysis of the coproporphyrinogen oxidase gene: two new mutations in DNA from Danish patients with hereditary coproporphyria

The knowledge of at least 21 different mutations and several polymorphisms in the coproporphyrinogen oxidase (CPO) gene demonstrates that the molecular basis of hereditary coproporphyria is heterogeneous. We developed a DGGE-based assay for the analysis of exons 2 to 7, including 14-96 nucleotides of the flanking intronic sequences of the CPO gene. To render it suitable for the clinical diagnostic laboratory, we designed the assay to allow use of identical PCR conditions and the same DGGE gel for analyses of all the regions. Using this assay, and subsequent sequencing of gene regions containing interallelic variations, two novel mutations in the CPO gene were identified: a missense mutation (607G-->A), leading to the substitution of an alanine with a threonine, and a nonsense mutation (1281G-->A), giving rise to a stop codon 28 codons upstream to the wild-type stop codon.     

Mechanism of drug resistance in Helicobacter pylori

Clarithromycin is one of the most important antibiotics for H. pylori eradication. However, 5-10% was reported to be resistant. It has been shown that one point mutation in the 23S rRNA gene is associated with resistance to clarithromycin. To detect H. pylori infection and the mutation simultaneously, we have designed PCR primers specific for H. pylori, and established assays of PCR-RFLP and PCR-preferential homo-duplex formation (PHFA). Using this assay, we can detect mixed infections with wild and mutant-strains. The prevalence of mutant infection increased through clarithromycin-based eradication. However, the existence of mutant strains had been confirmed before therapy in most cases who 'converted' to mutant after therapy. Metronidazole is also one of the most important antibiotics for eradication. However, 5-50% was reported to be resistant. It has been shown that rdx gene mutation is associated with resistance. It is reported that inactivation of the rdx gene is frequently, but not always, associated with resistance to metronidazole. Amoxicillin resistant strains were rare (1.2% in Japanese strains). It is reported that penicillin-binding protein might play a role in the resistance. By detecting of the resistance based on the molecular mechanism, patients can be treated with adequate antibiotics with information about resistance.     

Assessing heteroplasmic load in Leber's hereditary optic neuropathy mutation 3460G->A/MT-ND1 with a real-time PCR quantitative approach

To quantify the amount of the 3460G-->A/ND1 point mutation responsible for Leber's hereditary optic neuropathy, we developed a quantitative real-time polymerase chain reaction method based on the SYBR Green assay and a new approach using the TaqMan assay. Both methods were based on the amplification refractory mutation system, comparing the heteroplasmic load quantified by restriction fragment length polymorphism in 15 Leber's hereditary optic neuropathy family members, with the results obtained using quantitative real-time polymerase chain reaction methods. The comparative evaluation of mitochondrial DNA (mtDNA) heteroplasmy from blood samples showed significant correlation between restriction fragment length polymorphism analysis, real-time SYBR Green assay, and TaqMan assay. We validated the last method by measuring experimental samples composed by a known proportion of cloned plasmids containing either the wild-type or mutant sequence, giving a correlation coefficient of 0.999 (P < 0.0001). The real-time amplification refractory mutation system polymerase chain reaction by TaqMan assay provides a rapid, reliable, sensitive, reproducible, and one-step quantitative method to detect heteroplasmic mutant mtDNA. This method allows the quantitation of a broad range of mutational load (up to 100%, down to 0.01%) on the basis of in vitro calibration, thus rendering the TaqMan assay suitable for the diagnostic analysis of heteroplasmic load in mtDNA-related disorders.     

Rapid detection of ethambutol-resistant Mycobacterium tuberculosis strains by PCR-RFLP targeting embB codons 306 and 497 and iniA codon 501 mutations

Mutations at embB gene codons 306 and 497 and iniA gene codon 501 occur frequently in ethambutol (EMB)-resistant Mycobacterium tuberculosis strains worldwide. The identification of these mutations in resistant strains has been achieved by labor-intensive DNA sequencing or by tedious amplification protocols followed by restriction endonuclease digestion. In this report, we describe PCR-restriction fragment length polymorphism (RFLP)-based methods for determining substitutions at embB codons 306 and 497 and iniA codon 501 directly in BACTEC cultures of M. tuberculosis isolates. The wild-type and mutant alleles are revealed by easily interpretable and different RFLP patterns. The methods optimized initially on reference strains were tested directly on BACTEC cultures of 25 randomly selected clinical M. tuberculosis isolates, seven of which were determined to contain EMB-resistant strains by phenotypic drug susceptibility testing. The PCR-RFLP methods identified mutations in four of seven EMB-resistant strains with three isolates containing mutated embB codon 306 and one isolate containing mutated embB codon 497. The results of PCR-RFLP were confirmed by DNA sequencing. The worldwide prevalence figures for mutations at embB codons 306 and 497 and iniA codon 501 suggest that nearly half of EMB-resistant M. tuberculosis strains could be identified within one working day even in developing countries equipped with simple PCR technology instead of weeks required for phenotypic drug susceptibility testing. Further, since EMB resistance is also associated with multiple-drug resistance from some geographical locations, detection of EMB resistance may also lead to rapid identification of multidrug-resistant strains of M. tuberculosis.     

Rapid detection of K-ras mutations in bile by peptide nucleic acid-mediated PCR clamping and melting curve analysis: comparison with restriction fragment length polymorphism analysis

BACKGROUND: Current methods for detection of K-ras gene mutations are time-consuming. We aimed to develop a one-step PCR technique using fluorescent hybridization probes and competing peptide nucleic acid oligomers to detect K-ras mutations in bile and to compare the efficacy with restriction fragment length polymorphism (RFLP) analysis. METHODS: Bile samples were obtained from 116 patients with biliary obstruction, including gallstones (n = 64), benign biliary strictures (n = 6), pancreatic cancer (n = 20), and cholangiocarcinoma (n = 26). The DNA was extracted and subjected to K-ras mutation analysis by real-time PCR and RFLP analysis. Mutations were confirmed by direct sequencing. The sensitivity and specificity were calculated according to the clinical results. RESULTS: The analysis time for real-time PCR was <1 h, whereas RFLP analysis took more than 2 days. With the sensor probe designed for the GAT (G12D) mutant in codon 12 of the K-ras gene, the real-time PCR method also detected the GTT (G12V) mutant. In contrast, a specific sensor probe for the TGT (G12C) mutant detected GAT (G12D), AGT (G12S), and GTT (G12V) mutants in addition to the TGT mutant. The real-time PCR assay allowed the detection of mutation in a 3000-fold excess of wild-type bile DNA. In bile, K-ras codon 12 mutations were detected in 16 of 46 malignant cases by real-time PCR with the TGT probe and 15 by RFLP analysis. All benign cases were wild type. CONCLUSION: Real-time PCR with a cysteine-specific (TGT) sensor probe can rapidly detect K-ras gene mutations in bile and diagnose malignant biliary obstruction with high specificity.     

Rapid semiquantitative real-time PCR for the detection of human cytomegalovirus UL97 mutations conferring ganciclovir resistance

BACKGROUND: The development of infections with ganciclovir (GCV)-resistant human cytomegalovirus (HCMV) remains a serious problem in recipients of stem cell or organ transplants. Nearly all GCV-resistant clinical isolates have mutations in the viral UL97 gene. The rapid detection of GCV-resistant HCMV infections is necessary and the relative proportions of wild-type and mutant strains are predictive for the efficiency of antiviral therapy. To date, genotypical resistance screening has been limited to restriction fragment length polymorphism (RFLP) and sequencing analyses. Here, we present a comprehensive real-time PCR approach for the detection of most frequent mutations in the UL97 gene associated with GCV resistance. METHODS: The laboratory strains AD169 and Towne, different wild-type isolates and plasmids constructed by site-directed mutagenesis and overlap extension with specific point-mutations in the UL97 gene were analysed by LightCycler PCR and compared with UL97 RFLP and sequencing analyses. RESULTS: A new and comprehensive set of LightCycler PCRs was created using specific hybridization probes with melting-point analysis for the relevant codons 594, 595, 603 and 607. Different wild-type isolates and plasmids containing specific UL97 mutations conferring GCV resistance were investigated in the real-time PCR assay. Total processing time was 80 min per assay, whereas combinations of RFLP and sequencing needed at least 3-4 days. Proportions of co-existing wild-type and mutant strains in mixed viral populations can be obtained. CONCLUSIONS: We established a rapid real-time PCR approach for the detection of most frequent HCMV UL97 mutations associated with GCV resistance. Moreover, the method allows semiquantitative differentiation of the proportions of co-existing wild-type and mutant strains. This approach represents a new alternative for laborious RFLP analysis.     

Development of real-time PCR assays for the quantitative detection of Epstein-Barr virus and cytomegalovirus,comparison of TaqMan probes,and molecular beacons

Human Epstein-Barr virus (EBV) and cytomegalovirus (CMV) can cause serious complications in immunocompromised patients. Rapid diagnosis of EBV and CMV infection is critical in the management of the disease so that anti-viral therapy can be started early. Here we describe the development of real-time PCR assays using TaqMan probes and molecular beacons and compare the performance of both assays with a well-established, validated, gel-based PCR method for the quantification of EBV and CMV in patients’ samples. The TaqMan and molecular beacon assays were linear between 10 to 10⁷ viral genomes/reaction. Both assays generated calibration curves with strong correlation and low intra-assay and interassay variation. Results of EBV and CMV viral load determination inpatient samples obtained by the gel-based and real-time PCR were very similar. The real-time PCR assays showed increases in viral load before clinical measures of viral disease and decreases in viral load during anti-viral therapy in two of six pediatric patients. The data indicate that these TaqMan and molecular beacon approaches are accurate, rapid, and reliable assays for the diagnosis and monitoring of EBV and CMV infections in patients.     

Detection of gyrA and gyrB mutations in Clostridium difficile isolates by real-time PCR

Fluoroquinolone (FQ)-resistance in Clostridium difficile has been associated with mutations in the quinolone-resistance determining region (QRDR) of gyr genes. In particular, the majority of resistant clinical isolates show mutations in codon 82 of gyrA or in codon 426 of gyrB. A real-time PCR method was developed to identify these mutations in FQ-resistant C. difficile strains. Twenty-one clinical isolates, selected as representative of the different gyr alleles known up to date, and 20 clinical isolates with unknown behavior towards FQs were used to validate the method. Each mutation was detected by real-time amplification followed by hybridization with two fluorescent probes designed with the sequence complementary to the wild-type sequences of gyr genes. The melting peak analysis of the probe-PCR product hybrid was performed on a LightCycler (Roche Diagnostic). Single and multiplex assays were performed with the same reaction conditions. In both cases, isolates showing mutations in gyr sequences had a well distinguished T_m compared to that of isolates showing wild-type genes or silent mutated codons in the nucleotide region covered by probes. The results obtained indicate that this real-time PCR assay is a rapid, reproducible and accurate screening method of the predominant mutations determining FQ-resistance in C. difficile strains.     

Early detection of plasma cytomegalovirus DNA by real-time PCR after allogeneic hematopoietic stem cell transplantation

Cytomegalovirus (CMV) infection is an important cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Therefore, preemptive ganciclovir therapy based on early detection of CMV reactivation is widely used to prevent CMV disease. Real-time polymerase chain reaction (PCR) has been widely used for monitoring CMV reactivation as well as the antigenemia assay that detects CMV structural phosphoprotein with a molecular weight of 65,000 (pp65). We developed a real-time PCR assay system for CMV based on a double-stranded DNA-specific dye, SYBR Green I, and quantified DNA, which was extracted automatically from plasma. This real-time PCR assay and the pp65 antigenemia assay were compared in parallel with 357 blood samples obtained from 64 patients who underwent allogeneic hematopoietic stem cell transplantation (allo-HSCT). Real-time PCR assay results correlated with those of the pp65 antigenemia assay (p < 0.0001). It is noteworthy that the detection of CMV DNA by PCR preceded the first positive antigenemia by 14 days. In this study, 10 of 64 patients developed CMV disease. The antigenemia assay detected CMV reactivation earlier than the development of CMV disease only in four of 10 patients. In contrast, our real-time PCR detected CMV-DNA before the development of CMV diseases in eight of 10 patients. The real-time PCR with SYBR Green I as a detection signal is simple and readily performed, and may be a useful system for early detection of CMV reactivation after allo-HSCT.     

Rapid screening assay for KRAS mutations by the modified smart amplification process

Previously, the smart amplification process version 2 (SMAP-2) was developed to detect mutations from tissue and in crude cell lysates and has been used for rapid diagnosis of specific somatic mutations with single-nucleotide precision. The purpose of this study was to develop a rapid and practical method to detect cancer and metastasis in specimens using the SMAP-2 assay. We developed modified SMAP-2 assays that enabled detection of any change in a single codon using a single assay. Rapid SMAP-2 screening assays are suitable for routine clinical identification of critical amino acid substitutions such as codon 12 mutations in KRAS. Primers bracketing the first two nucleotides of KRAS codon 12 were designed so that all possible alleles would be amplified by the SMAP-2 assay. In combination with the peptide nucleic acid (PNA) with exact homology to the wild-type allele, our assay amplified all mutant alleles except for the wild-type sequence. With this new assay design (termed PNA-clamp SMAP-2), we could detect KRAS mutations within 60 minutes, including sample preparation. We compared results from PNA-clamp SMAP-2 assay, polymerase chain reaction-restriction fragment length polymorphism, and direct sequencing of clinical samples from pancreatic cancer patients and demonstrated perfect concordance. The PNA-clamp SMAP-2 method is a rapid, simple, and highly sensitive detection assay for cancer mutations.     

Short PNA molecular beacons for real-time PCR allelic discrimination of single nucleotide polymorphisms

The typing of a single nucleotide polymorphism with DNA probes is sometimes problematic because of the limited discriminating power of long DNA probes. As an alternative to existing assays, we have developed a real-time PCR assay for the genotyping of single nucleotide polymorphisms using short peptide nucleic acid (PNA) molecular beacons. A single nucleotide polymorphism in exon 6 of the XPD gene was chosen as the model system. The genotyping experiments were performed in the ABI 7700 using beacons labeled with either fluorescein or JOE, and in the Lightcycler using a fluorescein labeled beacon. QSY-7 was used as the quencher in all the beacons. The result of the genotyping was the same on both instruments and was in agreement with a previously performed RFLP genotyping of 79 samples. The length of PNA molecular beacons is significantly shorter than that of TaqMan or Lightcycler probes, making probe design and genotype discrimination easier.     

Non-PCR-dependent detection of the factor V Leiden mutation from genomic DNA using a homogeneous invader microtiter plate assay.

BACKGROUND: The goal of this study was to compare the Invader technology for the direct detection of the Factor V Leiden mutation from genomic DNA with a conventional polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. METHODS AND RESULTS: In the Invader assay, a specific upstream "invading" oligonucleotide and a partially overlapping downstream probe together form a specific structure when bound to complementary DNA template. This structure is recognized and cut at a specific site by the Cleavase enzyme, resulting in release of the 59 flap of the probe oligonucleotide. This fragment now serves as the "Invader" oligonucleotide with respect to synthetic secondary targets and secondary fluorescently labeled signal probes contained in the reaction mixture, resulting in specific cleavage of the secondary signal probe by the Cleavase enzyme. Fluorescence signal is generated when this secondary probe, labeled with dye molecules capable of fluorescence resonance energy transfer, is cleaved. Genomic DNAs isolated from peripheral blood buffy coats of patients previously tested for the Factor V Leiden mutation by PCR-RFLP were tested using an Invader assay specific for this mutation. In all 48 samples containing sufficient DNA for testing (30 normal, 16 heterozygous, 2 homozygous mutant), the genotype determined by the Invader assay was concordant with the PCR-RFLP results. CONCLUSION: These results indicate that a simple microtiter plate-based Invader assay can reliably genotype routine clinical patient samples for the Factor V Leiden point mutation without the need for PCR amplification, restriction enzyme digestion, or gel electrophoresis.