磁珠分离DNA技术检测HIV-1逆转录酶基因耐药性突变

HIV-1逆转录酶基因突变导致的耐药性严重影响了药物对病人的治疗效果,耐药性突变的检测对于指导合理用药具有重要意义。本工作发展了一种检测HIV-1逆转录酶基因耐药性突变的新方法。在两步MS-PCR方法的基础上,引入磁珠分离DNA技术并结合酶联免疫吸附检测方法(ELISA)检测逆转录酶基因耐药性突变T215F和Y181C。结果显示： MS-PCR结合磁珠分离技术和ELISA具有较高的灵敏度和特异性,阳性/阴性比值（P/N值）达到要求,突变型模板检测限为5%左右,耗时短且能进行高通量检测。无论在HIV-1耐药性突变还是其它的点突变检测中,该方法都具有较好的临床应用前景。     

Improved molecular diagnosis of dystrophin gene mutations using the multiplex ligation-dependent probe amplification method

Mutation detection in the DMD gene defective in Duchenne (DMD) and Becker muscular dystrophies (BMD) is complicated by the presence of 79 exons. The majority of recognized mutations are, however, copy number changes of individual exons, which traditionally have been identified by three common multiplex polymerase chain reaction (PCR) assays and/or Southern blotting. Here we report the use of the newly developed quantitative assay multiplex ligation-dependent probe amplification (MLPA) to determine the copy number of each of the 79 DMD exons in 182 males and 14 carrier females referred to our diagnostic facility on the clinical suspicion of DMD or BMD. The MLPA method confirmed all previously recognized mutations and identified an additional 28, including four point mutations. Also, the assay reliably identified 7 carrier females, which are usually not easily recognized. In our hands the method is highly reproducible, easy to handle, and has increased our mutation pick-up rate by a total of 33%.     

Detection of 100% of mutations in 124 individuals using a standard UV/Vis microplate reader: a novel concept for mutation scanning

We report the development of a simple and inexpensive assay for the detection of DNA polymorphisms and mutations that is based on the modification of mismatched bases by potassium permanganate. Unlike the chemical cleavage of mismatch assay, which also exploits the reactivity of potassium permanganate to detect genomic variants, the assay we describe here does not require a cleavage manipulation and therefore does not require expensive or toxic chemicals or a separation step, as mismatches are detected using direct optical methods in a microplate format. Studies with individual deoxynucleotides demonstrated that the reactivity with potassium permanganate resulted in a specific colour change. Furthermore, studies with synthetic oligonucleotide heteroduplexes demonstrated that this colour change phenomenon could be applied to detect mismatched bases spectrophotometrically. A collection of plasmids carrying single point mutations in the mouse β-globin promoter region was used as a model system to develop a functional mutation detection assay. Finally, the assay was validated as 100% effective in detecting mismatches in a blinded manner using DNA from patients previously screened for mutations using established techniques, such as sequencing, SSCP and denaturing high-performance liquid chromatography (DHPLC) analysis in DNA fragments up to 300 bp in length.     

Enzymatic mutation detection. Procedure for screening and mapping of mutations by immobilised endonuclease VII.

Endonuclease VII (endo VII) binds to non-pairing nucleotides in DNA. This served as the basis for the development of a mutation detection assay involving immobilised endo VII and heteroduplex DNAs made by hybridisation of mutant and wild type DNA. The use of microtiter plates allows screening of large numbers of samples. Localisation of mutations in positive samples can be done in the same assay in a second optional step.     

Non-phenotypic selection of N-methyl-N-nitrosourea-induced mutations in human cells.

The distribution of mutations in a particular gene as detected by a selective mutation assay could be affected by the structural properties of the target protein. To investigate this, we have analysed N-methyl-N-nitrosourea (MNU)-induced mutations in two restriction recognition sequences of a target gene for mutation analysis and compared these data with what previously observed in a phenotypic mutation assay. DNA base changes in the Ncil and EcoRV sites of the gpt gene maintained in human cells by a shuttle vector system were measured by restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) technique. After MNU-treatment of human cells, mutations were detected in the Ncil recognition sequence but not in the EcoRV site. DNA sequencing analysis revealed that all Ncil-resistant mutations were GC to AT transitions located over four bases of the Ncil recognition sequence. Only one of these mutations drastically affected the functionality of the GPT protein. The Ncil-resistant mutations were randomly distributed in both DNA strands of the gpt gene and were preferentially targeted at guanine residues flanked 5' by a guanine. Our results indicate that the structure of the GPT protein is the main contributor to the strand-specificity of MNU-induced mutations previously reported by using a phenotypic mutation assay. The potential use of the RFLP/PCR technique as a general tool for mutation detection is also discussed.     

New Cleavase Fragment Length Polymorphism method improves the mutation detection assay

Cleavase Fragment Length Polymorphism (CFLP) analysis is a convenient, accurate and highly sensitive method for the detection and localization of nucleic acid mutations. The assay is well suited for high-throughput screening and can be used to detect mutations in known and unknown nucleic acid samples. A recent improvement in the CFLP assay termed "temperature ramping" or "ramping" is reported here. This procedural improvement eliminates the need for time and temperature optimizations before the actual sample analysis. In this study, we compare the CFLP ramping procedure to the conventional CFLP optimization procedure and demonstrate equal, and in some cases improved, detection of point mutations. With ramping, CFLP reactions are identical for all DNA fragments analyzed, which allows for increased sample throughput, decreased assay time and lower overall cost.     

A Simple Allele-Specific PCR Assay for Detecting <Emphasis Type="Italic">FAD2</Emphasis> Alleles in Both A and B Genomes of the Cultivated Peanut for High-Oleate Trait Selection

In cultivated tetraploid peanut (2n = 4x = 40, AABB), the conversion of oleic acid to linoleic acid is mainly catalyzed by the Δ¹² fatty acid desaturase (FAD). Two homoeologous genes (FAD2A and FAD2B) encoding for the desaturase are located on the A and B genomes, respectively. Abolishing or reducing the desaturase activity by gene mutation can significantly increase the oleic acid/linoleic acid ratio. F435-derived high-oleate peanut cultivars contain two key mutations within the Δ¹² fatty acid desaturase gene which include a 1-bp substitution of G:C→A:T in the A genome and a 1-bp insertion of A:T in the B genome. Both of these mutations contribute to abolishing or reducing the desaturase activity, leading to accumulation of oleate versus linoleate. Currently, detection of FAD2 alleles can be achieved by a cleaved amplified polymorphic sequence marker for the A genome and a real-time polymerase chain reaction (PCR) marker for the B genome; however, detection of these key mutations has to use different assay platforms. Therefore, a simple PCR assay for detection of FAD2 alleles on both genomes was developed by designing allele-specific primers and altering PCR annealing temperatures. This assay was successfully used for detecting FAD2 alleles in peanut. Gas chromatography (GC) was used to determine fatty acid composition of PCR-assayed genotypes. The results from the PCR assay and GC analysis were consistent. This PCR assay is quick, reliable, economical, and easy to use. Implementation of this PCR assay will greatly enhance the efficiency of germplasm characterization and marker-assisted selection of high oleate in peanut.     

Rapid detection of resistance against rifampicin in isolates of Mycobacterium tuberculosis from Brazilian patients using a reverse-phase hybridization assay

The main objective of this study was to evaluate INNO-LiPA Rif.TB and to determine the frequency of mutations in rpoB in rifampicin-resistant Mycobacterium tuberculosis isolates of Brazilian tuberculosis patients. We used the reverse hybridization assay on 113 resistant and 15 sensitive clinical isolates of M. tuberculosis and on reference strains belonging to 37 different species. All MTB complex strains and none of the other strains reacted with the MTB complex-specific probe, meaning that the assay is 100% specific and 100% sensitive for detection of strains of the MTB complex. In 80 resistant strains, mutations causing S531L (n=55), H526Y (n=9), H526D (n=12) or D516V (n=9) were detected while in 30 strains, mutations were present but their exact nature was not determined by the assay (DeltaS patterns). All sensitive strains had the sensitive genotype while among resistant isolates, a sensitive genotype was obtained in three due to the absence of mutations in the hot spot region, demonstrating an assay accuracy of 97.6% for detection of drug susceptibility. In 10 resistant cultures, two or more mutations were detected and in five, mixed sensitive and resistant genotypes were observed. The sensitivity of the assay for detection of resistant organisms in a mixture with sensitive ones were 2% and 70%, respectively, considering the appearance and disappearance of the R2 and S2 bands. The sensitivity to detect heteroresistance is similar to that of the proportion method when a specific probe for the mutation is present but the performance of the assay in the patient population will depend on the frequency of mutation distribution.     

Universal DNA array detection of small insertions and deletions in BRCA1 and BRCA2

Array-based mutation detection methodology typically relies on direct hybridization of the fluorescently labeled query sequence to surface-bound oligonucleotide probes. These probes contain either small sequence variations or perfect-match sequence. The intensity of fluorescence bound to each oligonucleotide probe is intended to reveal which sequence is perfectly complementary to the query sequence. However, these approaches have not always been successful, especially for detection of small frameshift mutations. Here we describe a multiplex assay to detect small insertions and deletions by using a modified PCR to evenly amplify each amplicon (PCR/PCR), followed by ligase detection reaction (LDR). Mutations were identified by screening reaction products with a universal DNA microarray, which uncouples mutation detection from array hybridization and provides for high sensitivity. Using the three BRCA1 and BRCA2 founder mutations in the Ashkenazi Jewish population (BRCA1 185delAG; BRCA1 5382insC; BRCA2 6174delT) as a model system, the assay readily detected these mutations in multiplexed reactions. Our results demonstrate that universal microarray analysis of PCR/PCR/LDR products permits rapid identification of small insertion and deletion mutations in the context of both clinical diagnosis and population studies.     

Single-strand conformation polymorphism analysis by capillary and microchip electrophoresis: a fast, simple method for detection of common mutations in BRCA1 and BRCA2

As a result of intensive studies on hereditary breast and ovarian cancers, two breast cancer susceptibility genes, BRCA1 and BRCA2, have been identified. In each gene, a small number of specific mutations have been found at relatively high frequency in certain ethnic populations. The mutations, 185delAG and 5382insC in BRCA1 and 6174delT in BRCA2, have been identified as common mutations in the Ashkenazi Jewish population, with a combined frequency of 2.0 to 2.5%. Women who have one of the above three common mutations are at a high risk of developing breast or ovarian cancer. Consequently, accurate and cost-effective detection of these three mutations may have important implications for risk assessment in susceptible women and men. In this report, we describe a fast and simple capillary electrophoresis (CE)-based method using a polymer network for screening the three common mutations in BRCA1 and BRCA2. Fluorescent dye-labeled primers (6-FAM-tagged) were used to amplify three DNA fragments of 258, 296, and 201 bp for detection of the 185delAG, 5382insC, and 6174delT mutations, respectively. After the PCR products were denatured, a single-strand conformation polymorphism (SSCP) profile could be obtained for each mutation in less than 10 min by CE in a polymer network. We demonstrate the potential provided by translating this assay to the microchip format where the SSCP analysis is complete in 120 s, representing only a fraction of the reduction in analysis time that can be achieved with microchip technology. The speed and simplicity of the SSCP methodology for detection of these mutations make it attractive for use in the clinical diagnostic laboratory.     

The present status of higher plant bioassays for the detection of environmental mutagens

Higher plants provide valuable genetic assay systems for screening and monitoring environmental pollutants. They are now recognized as excellent indicators of cytogenetic and mutagenic effects of environmental chemicals and are applicable for the detection of environmental mutagens both indoor and outdoor. Comparisons between plant and nonplant genetic assay systems indicate that higher plant genetic assays have a high sensitivity (i.e. few false negatives). Two assays which are considered ideal for in situ monitoring and testing of airborne and aqueous mutagenic agents are the Tradescantia stamen hair assay for mutations and the Tradescantia micronucleus assay for chromosome aberrations. Both assays can be used for in vivo and in vitro testing. Other higher plant gentoxicity assys which have a large number of genetic markers and/or data base and are also highly suitable for testing for genotoxic agents include Arabidopsis thaliana, Allium cepa, Hordeum vulgare, Vicia faba, and Zea mays. Since higher plant systems are now recognized as excellent indicators of the cytotoxic, cytogenetic, and mutagenic effects of environmental chemicals and have unique advantages for in situ monitoring and screening it is recommended that higher plant systems be accepted by regulatory authorities as an alternative first-tier assay system for the detection of possible genetic damage resulting from pollution or the use of environmental chemicals. The results from higher platn genetic assays could meke a significant contribution in protecting the public from agents that can cause mutation and cancer. The advantages possessed by higher plant genetic assays, which are inexpensive and easy to handle, make them ideal for use by scientists in developing countries.     

The Use of a Two-Tiered Testing Strategy for the Simultaneous Detection of Small EGFR Mutations and EGFR Amplification in Lung Cancer

Lung cancer is the leading cause of cancer-related death worldwide. Recent progress in lung cancer diagnosis and treatment has been achieved due to a better understanding the molecular mechanisms of the disease and the identification of biomarkers that allow more specific cancer treatments. One of the best known examples of personalized therapy is the use of tyrosine kinase inhibitors, such as gefitinib and erlotinib, for the successful treatment of non-small-cell lung cancer patients selected based on the specific EGFR mutations. Therefore, the reliable detection of mutations is critical for the application of appropriate therapy. In this study, we tested a two-tiered mutation detection strategy using real-time PCR assays as a well-validated high-sensitivity method and multiplex ligation-dependent probe amplification (MLPA)-based EGFRmut+ assay as a second-tier standard-sensitivity method. One additional advantage of the applied MLPA method is that it allows the simultaneous detection of EGFR mutations and copy-number alterations (i.e., amplifications) in EGFR, MET and ERBB2. Our analysis showed high concordance between these two methods. With the use of this two-tier strategy, we reliably determined the frequency of EGFR mutations and EGFR, MET and ERBB2 amplifications in over 200 lung cancer samples. Additionally, taking advantage of simultaneous copy number and small mutation analyses, we showed a very strong correlation between EGFR mutations and EGFR amplifications and a mutual exclusiveness of EGFR mutations/amplifications with MET and ERBB2 amplifications. Our results proved the reliability and usefulness of the two-tiered EGFR testing strategy.     

重复引物PCR技术在超大片段动态突变疾病基因检测中的应用进展

动态突变疾病是指基因编码区或非编码区发生核苷酸重复序列异常扩增所导致的一类遗传性疾病。发生于非翻译区的动态突变常常伴有超大片段重复序列,应用普通PCR法无法对该片段进行扩增,而传统的Southern blot等技术费时费力,无法应用于临床基因诊断。在此背景下,重复引物PCR技术应运而生。本文将分别阐述重复引物PCR技术在强直性肌营养不良症、Friedreich共济失调、脊髓小脑性共济失调10型及C9orf72基因突变引起的额颞叶痴呆或肌萎缩侧索硬化等疾病基因检测中的应用进展。     

Benchmarking and optimization of a high-throughput sequencing based method for transgene sequence variant analysis in biotherapeutic cell line development

In recent years, High-Throughput Sequencing (HTS) based methods to detect mutations in biotherapeutic transgene products have become a key quality step deployed during the development of manufacturing cell line clones. Previously we reported on a higher throughput, rapid mutation detection method based on amplicon sequencing (targeting transgene RNA) and detailed its implementation to facilitate cell line clone selection. By gaining experience with our assay in a diverse set of cell line development programs, we improved the computational analysis as well as experimental protocols. Here we report on these improvements as well as on a comprehensive benchmarking of our assay. We evaluated assay performance by mixing amplicon samples of a verified mutated antibody clone with a non-mutated antibody clone to generate spike-in mutations from ∼60% down to ∼0.3% frequencies. We subsequently tested the effect of 16 different sample and HTS library preparation protocols on the assay's ability to quantify mutations and on the occurrence of false-positive background error mutations (artifacts). Our evaluation confirmed assay robustness, established a high confidence limit of detection of ∼0.6%, and identified protocols that reduce error levels thereby significantly reducing a source of false positives that bottlenecked the identification of low-level true mutations.     

MutS as a tool for mutation detection

MutS, a DNA mismatch-binding protein, seems to be a promising tool for mutation detection. We present three MutS based approaches to the detection of point mutations: DNA retardation, protection of mismatched DNA against exonuclease digestion, and chimeric MutS proteins. DNA retardation in polyacrylamide gels stained with SYBR-Gold allows mutation detection using 1-3 microg of Thermus thermophilus his6-MutS protein and 50-200 ng of a PCR product. The method enables the search for a broad range of mutations: from single up to several nucleotide, as mutations over three nucleotides could be detected in electrophoresis without MutS, due to the mobility shift caused by large insertion/deletion loops in heteroduplex DNA. The binding of DNA mismatches by MutS protects the complexed DNA against exonuclease digestion. The direct addition of the fluorescent dye, SYBR-Gold, allows mutation detection in a single-tube assay. The limited efficiency of T4 DNA polymerase as an exonuclease hampers the application of the method in practice. The assay required 300-400 ng of PCR products in the range of 200-700 bp and 1-3 microg of MutS. MutS binding to mismatched DNA immobilised on a solid phase can be observed thanks to the activity of a reporter domain linked to MutS. We obtained chimeric bifunctional proteins consisting of T. thermophilus MutS and reporter domains, like beta-galactosidase or GFP. Very low detection limits for beta-galactosidase could theoretically enable mutation detection not only by the examination of PCR products, but even of genomic DNA.     

Detection of DNA mutations by fluorescence resonance energy transfer-based preferential homoduplex formation assay

Molecularly targeted agents for cancer therapy are recognized as being effective and are gaining in popularity. However, the efficacy of the agents depends on the status of the targeted molecule such as the number of molecules expressed, activity, and mutation. Therefore, the use of companion diagnostics for investigating the status of the targeted molecule prior to therapy is highly important. We developed a simple and cost-effective somatic mutation detection method called the fluorescence resonance energy transfer-based preferential homoduplex formation assay (FRET–PHFA). By using double-stranded labeled DNA and fluorescence measurement with thermal control, this method provides higher reproducibility, easier handling, less risk for contamination, shorter assay time (only ∼15 min), and less cost compared with conventional PHFA. Here we report the evaluation of FRET–PHFA on the detection of multiallelic KRAS mutations in codons 12 and 13 compared with the TheraScreen clinical diagnostics kit. We found that FRET–PHFA detected KRAS mutations (1.25–50%) from all cell line DNA titration samples.     

Adaptation of conformation-sensitive gel electrophoresis to an ALFexpress DNA sequencer to screen BRCA1 mutations

Conformation-sensitive gel electrophoresis (CSGE) has been introduced as the most reliable method for the screening of large and multi-exon genes because of its simplicity, sensitivity and specificity. Based on heteroduplex formation and with the use of mildly denaturing solvents, it allows detection of single-base mutations with accuracy. This is important in genes such as BRCA1 and BRCA2, in which alterations span the entire gene. We have adapted the CSGE assay to a fluorescent platform--a DNA sequencer one-color technology--that reduces the time involved and enhances resolving power for the complete scanning of the BRCA genes. Electrophoresis has high sensitivity and is performed in less than three hours, and the gel does not require staining with ethidium bromide. Eighteen single-base and six frameshift mutations in the BRCA1 gene were analyzed. We compared the manual and fluorescent CSGE methods, and all mutations were detected with accuracy.     

Sequencing-based detection of low-frequency human immunodeficiency virus type 1 drug-resistant mutants by an RNA/DNA heteroduplex generator-tracking assay

Drug-resistant viruses may be present as minority variants during early treatment failures or following discontinuation of failed antiretroviral regimens. A limitation of the traditional direct PCR population sequencing method is its inability to detect human immunodeficiency virus type 1 (HIV-1) variants present at frequencies lower than 20%. A drug resistance genotyping assay based on the isolation and DNA sequencing of minority HIV protease variants is presented here. A multiple-codon-specific heteroduplex generator probe was constructed to improve the separation of HIV protease genes varying in sequence at 12 codons associated with resistance to protease inhibitors. Using an RNA molecule as probe allowed the simple sequencing of protease variants isolated as RNA/DNA heteroduplexes with different electrophoretic mobilities. The protease gene RNA heteroduplex generator-tracking assay (RNA-HTA) was tested on plasma quasispecies from 21 HIV-1-infected persons in whom one or more protease resistance mutations emerged during therapy or following initiation of salvage regimens. In 11 of 21 cases, RNA-HTA testing of virus from the first episode of virologic failure identified protease resistance mutations not seen by population-based PCR sequencing. In 8 of these 11 cases, all of the low-frequency drug resistance mutations detected exclusively by RNA-HTA during the first episode became detectable by population-based PCR sequencing at the later time point. Distinct sets of protease mutations could be linked on different genomes in patients with high-frequency protease gene lineages. The enhanced detection of minority drug resistance variants using a sequencing-based assay may improve the efficacy of genotype-assisted salvage therapies.     

A real-time PCR assay for detection of isoniazid resistance in Mycobacterium tuberculosis clinical isolates

A real-time PCR genotypic assay was developed for the detection of isoniazid (INH) resistance in Mycobacterium tuberculosis. The assay detects mutations C(-15)T and, possibly, G(-24)T in the regulatory region of the inhA gene and proved as sensitive and specific as nucleotide sequencing in all the clinical isolates tested. Our assays mapped the mutations efficiently in 10 out of 35 resistant isolates, thereby covering 29% of all resistant strains.