Geographical clusters of dengue virus type 2 isolates based on analysis of infected cell RNA by the chemical cleavage at mismatch method.

Genetic variation in 12 strains of dengue virus type 2, isolated from several epidemic areas in different years, was studied by chemical cleavage at mismatched cytosine in DNA:RNA heteroduplexes. End-labelled cDNA probes derived from the E and NS2A genes of the New Guinea C strain were hybridized to total RNA extracted from cells infected by individual isolates. Following modification of mismatched cytosine by hydroxylamine and nucleic acid strand cleavage by piperidine, the resulting fragments of radiolabelled probe were analysed by electrophoresis and autoradiography. The patterns of bands generated corresponded to the geographical groupings of the isolates. Thus this method is suitable in epidemiological studies for rapidly surveying a large number of isolates for genetic variation in a particular gene of interest.     

Diagnosis of multiple endocrine neoplasia [MEN] 2A, 2B and familial medullary thyroid cancer [FMTC] by multiplex PCR and heteroduplex analyses of RET proto-oncogene mutations

Multiple endocrine neoplasia type 2 [MEN 2] is an autosomal dominant cancer syndrome with two subtypes, 2A and 2B. MEN 2A and medullary thyroid cancer [MTC] are caused by >25 different point mutations in exons 10, 11, and 13 of the RET proto-oncogene, whereas MEN 2B is caused by a single exon 16-point mutation. Various molecular methods have been used to identify the different mutations, including DNA sequencing, restriction enzymatic analyses, chemical cleavage mismatch, Single Stranded Conformational Polymorphism [SSCP], and Denaturing Gradient Gel Electrophoresis [DGGE]. These techniques, although useful and accurate, are labor intensive and some involve the use of radioactivity. We have developed a multiplex PCR assay simultaneously to amplify exons 10, 11, and 13 of the RET proto-oncogene. The multiplex PCR product is then analyzed on a modified Mutation Detection Enhancement [MDE] matrix for heteroduplex identification and visualized with ethidium bromide. Distinct heteroduplexes were detected for each known RET proto-oncogene mutation available in our laboratory (nine in exon 10, five in exon 11, one in exon 13, and the single exon 16 mutation). Presymptomatic DNA diagnosis of MEN 2 is essential since pentagastrin-stimulated calcitonin studies can occasionally produce false positive results and lead to unnecessary thyroidectomies. Prophylactic thyroidectomy is recommended by age 5 or 6 once a mutation is identified in a patient, since penetrance is very high. MDE heteroduplex detection provides a quick, efficient, and inexpensive method of screening for RET mutations in MTC patients with unknown mutations, or for presymptomatic diagnosis in individuals at risk for inheriting a known RET mutation. Confirmation of the specific mutation can be achieved by restriction enzymatic digestion (if feasible) or by DNA sequencing. © 1996 Wiley-Liss, Inc.     

Rapid chemical mapping of dengue virus variability using RNA isolated directly from cells

Osmium tetroxide and hydroxylamine (in combination with piperidine) have previously been shown to cleave mismatched T and C bases, respectively, in DNA.DNA heteroduplexes. In this work we report that mismatched T and C bases were similarly cleaved in DNA.RNA heteroduplexes of nucleic acids derived from different strains of dengue virus type 2. Further, some matched T or C bases one or two bases from mismatches were also chemically reactive and thus cleavable as detected by minor bands. Cleavages both at and near mismatches combined to generate a simply obtained pattern of difference between virus strains that could be used as a fingerprint of a given virus relative to another. The patterns obtained using viral RNA of one strain hybridized with the cDNA of another were similar for RNA prepared from purified virions and for total RNA extracted from infected cells. Use of probes of both senses should detect all differences. Two sequenced (NGC and PUO-218) and one unsequenced (D80-100) strains of virus were compared in these studies. The analyses allowed proof reading of the differences between NGC and PUO-218, ascertained from nucleotide sequencing, and demonstrated that D80-100 is more similar to PUO-218 than to NGC.     

DNA Ladder的制备

背景：目前,制备DNA分子量标准的方法主要有2种,一种是用限制性内切酶消化某种DNA,另一种是利用PCR扩增,2种方法各有优缺点。在前期采用PCR技术在前期扩增100~500 bp片段的基础上,实验室又成功扩增出600~1 000 bp片段,PCR产物经过纯化,混匀,制备的DNA Ladder,结果制备DNA Ladder的条带清晰,易于识别,可完全与公司商品化的DNA Ladder 相比,完全可用于分子生物学实验。 目的：利用PCR扩增技术制备DNA分子量标准参照物。 方法：自行构建了一种特殊适宜扩增的质粒pUC-DNA,根据pUC-DNA的基因序列,利用primer5.0设计能特异扩增100~ 1 000 bp 的PCR引物。PCR扩增出100~1 000 bp大小的DNA片段,在2%琼脂糖凝胶中电泳观察结果。用凝胶回收试剂盒回收目的PCR 产物,测序结果与pUC-DNA上基因序列进行序列比对,Blast进行同源性分析。将PCR产物用酚/氯仿抽提,乙醇沉淀,按比例混匀,即可使用。 结果与结论：利用PCR技术能够成功扩增出100~1 000 bp 条带,片段大小与预期结果相符,片段序列与GenBank序列完全一致,利用回收片段制备的DNA Ladder 条带清晰,可与同类产品相比。     

Xeroderma pigmentosum variant: complementary molecular approaches to detect a 13 base pair deletion in the DNA polymerase eta gene

Deficiencies of DNA polymerase eta—an enzyme mediating replication past UV-induced DNA damage—predispose individuals to xeroderma pigmentosum variant (XPV) and result in a high incidence of skin cancers. We designed, developed and assessed several complementary molecular approaches to detect a genetically inherited deletion within DNA polymerase eta. RNA was reverse transcribed from XPV fibroblasts and from normal human cells, and standard polymerase chain reaction (PCR) was conducted on the cDNA targeting a region with a 13 base pair deletion within the polymerase eta gene. PCR products were subjected to restriction fragment length polymorphism (RFLP) analysis and cycle DNA sequencing. The deletion was found to eliminate a BsrGI restriction site and affected the number of resultant fragments visualized after gel electrophoresis. Cycle sequencing of polymerase eta-specific amplicons from XPV and normal cells provided a second approach for detecting the mutation. Additionally, the use of a fluorescent nucleic acid dye—EvaGreen—in real-time PCR and melt curve analysis distinguished normal and XPV patient-derived amplicons as well as heteroduplexes that represent heterozygotic carriers without the need for high resolution melt analysis-compatible software. Our approaches are easily adaptable by diagnostic laboratories that screen for or verify genetically inherited disorders and identify carriers of a defective gene.     

Simultaneous genotyping for all three known structural mutations in the human mannose-binding lectin gene

We describe a rapid and simple method for genotyping the three known structural mutations within exon 1 of the mannan-binding lectin (MBL) gene. A PCR-amplifiable synthetic DNA (Universal Heteroduplex Generator) was annealed to genomic PCR product from exon 1 to generate unique DNA heteroduplexes for each mutation. Heteroduplexes were then resolved by non-denaturing polyacrylamide gel electrophoresis. The technique was initially validated with previously typed samples and then applied to previously untyped samples with the results confirmed by DNA sequencing. © 1997 Wiley-Liss, Inc.     

A simple method for screening bacterial colonies for mutagenized sites in plasmid DNA.

Because of the multiple-step process that is involved in the detection of mutagenized restriction enzyme sites in plasmid DNA, a simple and accurate method was developed to analyse the plasmid DNA of site-directed mutagenesis experiments from bacterial colonies. The desired mutated part is located between the Eco RI restriction site on pUC19. Two mutagenic primers were designed to replace only one nucleotide on segments A and B of the bi-segmented genome of infectious bursal disease virus (IBDV). Two restriction sites were created for those mutations in each segment, Fsp I and Dra I, respectively. Following a protocol from the site-directed mutagenesis kit, the mutated plasmids were used to transform, and were propagated and maintained in DH5 alpha competent cells. Colonies were picked from the master plate, and used as DNA template for PCR. The PCR technique included the design of two pairs of primers, one for each segment, which were to amplify a region up to 1000 bp. Samples were pre-incubated for 3 min at 94 degrees C to induce bacterial lysis before starting the nucleic acid amplification. The PCR products 918 bp from segment A and 650 bp from segment B were digested with Fsp I and Dra I at 37 degrees C for 1 h. Products were resolved on 0.9% agarose gel which contained ethidium bromide. This method is simpler, faster and more accurate than the traditional method of mini-prep plasmid isolation and colony blot hybridization to identify the mutated plasmids.     

Extraction of DNA from oral cytological samples by scraping and smear method suitable for restriction site mutation analysis: a pilot study

The restriction site mutation assay (RSM) can be used to measure base changes which occur in the DNA coding for bacterial restriction enzymes. The aim of this study was to investigate whether DNA, of sufficient quantity and quality for analysis by RSM, could be extracted from cells collected from precancerous lesions using a cytological smear technique. Six smears were collected from each of five lesions of leukoplakia displaying a variety of clinical appearances. Three methods for the extraction of DNA were compared. The commercial extraction method was shown to be most convenient and reproducible, routinely providing 1-5 microg of DNA per sample. Cell populations collected by a cytological smear technique can provide DNA of sufficient quantity and quality for analysis of RSM.     

Restriction site mutation analysis, a proposed methodology for the detection and study of DNA base changes following mutagen exposure

We propose a methodology for the detection of DNA base changes in restriction enzyme recognition sites. The restriction site mutation (RSM) technique is based upon the detection of DNA sequences resistant to the cutting action of specific restriction enzymes and the amplification of these resistant sequences using the polymerase chain reaction. As outlined, the RSM method has the potential for use in the study of induced base changes in any species, tissue and genes of known DNA sequence for which unique DNA primers are available, and which contains a number of unique restriction enzyme recognition sites.     

Appearances can be deceptive: an APC 1893del4 mutation with unusual properities. Mutations in brief no. 171. Online

During a systematic search for germ-line APC mutations causative of familial adenomatous polyposis, we discovered what appeared to be an insertion mutation while simply checking exon 14PCR products by agarose gel electrophoresis (AGE). On AGE, exon 14PCR product from the known affected member of this family gave two bands: one of normal length, the other retarded on the gel equivalent to an increase in length of some 20-25 bp. Direct sequencing of DNA purified from the two bands gave identical results, and was consistent with amplification from the same two alleles: one wild-type, and the other having an 1893del4 mutation. This suggested that the normal length band on AGE consisted of DNA homoduplexes (normal:normal and mutant:mutant) and the retarded band consisted of DNA heteroduplexes (normal:mutant and mutant:normal). This hypothesis was tested by subjecting purified material from each of the two bands alone to a single cycle of heat denaturation and annealing, which showed that either band was equally capable of regenerating both bands. Because the anomalous migration of the heteroduplexes is observed in the presence of ethidium bromide, it implies that they have a cruciform of cruciform-like structure. This case illustrates the necessity to be aware of anomalous DNA migration and always sequence all putative mutations.     

Mapping of the single cleavage site in S13 replicative form DNA of Hemophilus influenzae (Hind III) restriction enzyme.

Bacteriophage S13 RF DNA is cleaved once by restriction enzyme Hind III. The site of the single cleavage has been mapped and the Hin d II cleavage map corrected.     

Discrimination of the ITS1 types of Fasciola spp. based on a PCR–RFLP method

Molecular characterization is important for discriminating Fasciola specimens having the deoxyribonucleic acid (DNA) sequences of Fasciola hepatica, Fasciola gigantica, and both Fasciola species, since three Fasciola forms coexist in Asian countries. We have developed a restriction fragment length polymorphism of amplified DNA (PCR–RFLP) of the nuclear ribosomal internal transcribed spacer 1 (ITS1) region in Fasciola species. The band patterns of the fragments digested with a restriction enzyme, Rsa I, were accurately distinguished among the three forms of Fasciola. Amplicons with the sequences of F. hepatica and F. gigantica were divided into fragments of about 360, 100, and 60 bp, and 360, 170, and 60 bp, respectively, and amplicons with the sequences of both Fasciola species yielded fragments of 360, 170, 100, and 60 bp. The results of PCR–RFLP completely coincided with those of sequence analysis, and thus PCR–RFLP is a useful technique for determining the ITS1 type in Fasciola species.     

Ligation of a primer at a mutation: a method to detect low level mutations in DNA

Detection of low frequency mutations following exposure to mutagens or during the early stages of cancer development is instrumental for risk assessment and molecular diagnosis. We present a sensitive new method to detect trace levels of DNA mutations induced within a large excess of wild-type sequences. The method is based on mutation-induced generation of new restriction enzyme recognition sites. A DNA sequence is amplified from genomic DNA or cDNA using a high fidelity polymerase. The purified PCR product is digested with a restriction enzyme that recognizes the newly generated restriction site, partially dephosphorylated and ligated with an oligonucleotide at the position of the mutation. The ligated oligonucleotide is then utilized in two rounds of PCR to amplify the mutated DNA but not the wild-type allele that contains no restriction site. An A-->T polymorphism in mRNA (tenascin gene, A(2366)-->T, Asn-->Ile) and a G-->A polymorphism in genomic DNA (Ku gene, G(74582)-->A, Val-->Ile), both of which generate a restriction site for the enzyme SAU3A1, demonstrate the application. Eleven patient samples pre-characterized for the G(74582)-->A polymorphism in the repair gene Ku are used to demonstrate the reliability of this approach. This technique quantitatively detects the Ku G-->A polymorphism at a mutant frequency of 1.6x10(-6) relative to the wild-type allele. Mutations in p53 that are frequently induced by mutagens can readily be detected using the present method. As an example, using a second enzyme BbvI, a mutation frequently encountered in human cancers (G(14154)-->A mutation, p53 codon 245, Arg-->Gln) was detected in patient samples. The process does not require radioactivity, utilizes established procedures and overcomes several factors known to produce false positives in RFLP-based assays. The present amplification via primer ligation at the mutation (APRIL-ATM) has potential applications in the detection of mutagen-generated genetic alterations, early detection of tumor marker mutations in bodily discharges and the diagnosis of minimal residual disease.     

DNA sequence analysis of hprt mutations in lymphocytes from Sprague-Dawley rats treated with 7,12-dimethylbenz[a]anthracene

Treatment of female Sprague-Dawley rats with the potent mammary gland carcinogen 7,12-dimethylbenz[a]anthracene (DMBA) results in the formation of DNA adducts with dG and dA and in the induction of 6-thioguanine-resistant (TG′) lymphocyte mutants. In this study, we have examined the types of mutations induced in TG′ lymphocytes from DMBA-treated rats. DNA from 263 TG′ lymphocyte clones was screened for mutations in exons 2, 3, and 8 of the hprt gene by polymerase chain reaction (PCR) amplification of the exons followed by heteroduplex analysis using denaturing gradient-gel electrophoresis. Twenty-five of the clones produced heteroduplexes in exon 2, 35 produced heteroduplexes in exon 3, and 36 produced heteroduplexes in exon 8. Direct sequence analysis of the heteroduplexes revealed 96 mutations, and at least 74 of these mutations were produced independently. Eighty-five of the total mutations were simple base pair (bp) substitutions, with A → T and G → T transversions being the predominant types. Seven mutations were deletions, three were complex bp substitutions, and one was an insertion. The results suggest that the types of mutations produced by DMBA in rat lymphocytes are specific to the DNA adducts produced by this compound. © 1996 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

EGFR activating mutations detected by different PCR techniques in Caucasian NSCLC patients with CNS metastases: short report

EGFR mutation testing has become an essential determination to decide treatment options for NSCLC. The mutation analysis is often conducted in samples with low percentage of tumour cells from primary tumour biopsies. There is very little evidence that samples from metastatic tissues are suitable for EGFR testing. We had evaluated the frequency of EGFR mutations with three highly sensitive PCR techniques in formalin-fixed, paraffin-embedded samples of 143 NSCLC patients with central nervous system (CNS) metastases. 32 corresponding primary tumours were also examined. We used PCR followed by DNA fragments length analysis (FLA), ASP–PCR and PNA–LNA PCR clamp techniques. We found 9 (6.29 %) EGFR gene mutations in CNS samples: 3 (2.1 %) in exon 19 and 6 (4.2 %) in exon 21. The full concordance between CNS metastases and primary tumour samples was observed. PCR followed by DNA–FLA and PNA–LNA PCR clamp were sensitive enough to detect exon 19 deletions. Two mutations in exon 21 were detected by ASP–PCR only, one L858R substitution was detected only by PNA–LNA PCR clamp. With respect to sensitivity, PCR followed by DNA–FLA achieved a level of detection of at least 10 % of mutated DNA for exon 19 deletion, as for ASP–PCR it was at least 5 % of mutated DNA for L858R substitution. Higher sensitivity of 1 % of mutated DNA was achieved by PNA–LNA PCR clamp technique for both mutations. The use of different methodological techniques authenticates the negative result of molecular tests.     

Mutations and DNA diagnoses of classical citrullinemia

Classical citrullinemia is an autosomal recessive disease caused by a genetic deficiency of argininosuccinate synthetase (ASS). We have previously identified 20 mutations in ASS mRNA of human classical citrullinemia and already established the DNA diagnosis of seven mutations as follows. By Southern blot analysis, each of the alleles with exon 5 or 6 deletion in mRNA appears to involve deletion of genomic DNA from this region. Five mutations involving R304W, G324S, IVS-6⁻² (ΔEx7), IVS-13⁺⁵ (ΔEx13), and Δ13bp Ex15&IVS-15 (ins37b/Ex15&16) are diagnosed by a combination of PCR (or modified PCR) and restriction enzyme digestion. It is important to identify the mutation in genomic DNA for prenatal diagnosis and carrier detection. In the present study, we report a novel missense mutation (R279Q) and a new abnormality in the ASS gene (Δ11bp/IVS-15). As three missense mutations (R272C, R279Q, and G280R) were found in exon 12, we isolated and sequenced the intron regions surrounding exon 12 to establish a DNA diagnostic test. Although a mutation with a deletion of the first seven bases in exon 16 of mRNA (Δ7b/Ex16) was found in both Japanese and American patients, the abnormality on the ASS gene was different between the Japanese allele (Δ11bp/IVS-15) and American allele (IVS-15⁻¹). The DNA diagnosis of 47 Japanese alleles with classical citrullinemia showed that the IVS-6⁻² and R304W mutations were found in 49% and 17% of the mutated alleles, respectively. We now have DNA diagnosis systems to detect 14 out of 22 mutations and are performing prenatal diagnosis and carrier detection using genomic DNA on classical citrullinemia. Hum Mutat 9:250–259, 1997. © 1997 Wiley-Liss, Inc.     

First case of missense mutation (LDH-H:R171P) in exon 4 of the lactate dehydrogenase gene detected in a Japanese patient

Complete deficiency of lactate dehydrogenase (LDH) subunit H was identified in a 41-year-old woman with paralysis of her left lower limb. The propositus had extremely low LDH activity and five of her family members had levels of LDH activity that ranged from lower than normal to normal level. A transversion mutation at codon 171 (CGC?CCC), resulting in an Arg?Pro substitution was identified in her DNA sequence. A new NruI restriction site was introduced into the polymerase chain reaction (PCR) product by PCR-primer introduced restriction analysis (PCR-PIRA) using a specific mismatched primer. Digestion with NruI revealed that the propositus and her mother were, respectively, homozygous and heterozygous for this mutation.     

DNA diagnosis of inherited metabolic diseases]

In recent years, dramatic advances in the applications of molecular genetic techniques have made it possible to identify molecular defects in man that account for inherited metabolic diseases. Thus molecular genetic techniques has rapidly expanded our ability to diagnose disease. DNA diagnosis includes prenatal and presymptomatic diagnosis as well as identification of carriers of many inherited metabolic disorders. Disorders that are always caused by a specific mutation can be diagnosed by direct detection of the mutation, while diagnosis of disorders with genetic heterogeneity or unknown genetic defect can be based on polymorphism in linkage analysis. DNA amplification by the PCR has made possible the rapid identification of DNA sequences variations. After PCR, mutations can be detected by various methods such as restriction enzyme analysis, amplification refractory mutation system, dot blot hybridization, chemical cleavage, or single strand conformational polymorphism. In addition, the PCR technique can be applied to polymorphism analysis. These methods have made DNA diagnosis quicker, easier, and cheaper.     

An update on conformation sensitive gel electrophoresis

Conformation-sensitive gel electrophoresis (CSGE) was developed as a method of heteroduplex analysis to screen large multi-exon genes for sequence variation. The novelty of the method was in the use of a non-proprietary acrylamide gel matrix that used 1,4-bis (acrolyl) piperazine (BAP) as a cross linker with ethylene glycol and formamide as mildly denaturing solvents. The denaturing environment enhances the conformation polymorphism present in DNA heteroduplexes containing variations as small as single nucleotide polymorphisms (SNPs). CSGE has also been adapted for use on a fluorescent platform (F-CSGE) that resulted in higher throughput and sensitivity. Variation in sensitivity of CSGE has been studied extensively. The results demonstrate that the nature of the mismatched base in a defined sequence context has the most profound effect on the conformation of the heteroduplex. Additionally, the size of the PCR product, as well as the location of the mismatch within the PCR product, are two important parameters that determine the resolution of the mismatch-containing heteroduplexes during CSGE. Like any other mutation scanning technique, CSGE can have limited resolution of two closely linked sequence variations. For specific genes, like BRCA1 and BRCA2 where multiple SNPs are present in the coding sequence, each CSGE shift has to be sequenced to define the exact nature of the sequence change. In conclusion, CSGE scanning provides a powerful, cost-efficient way to scan genes with high sensitivity and specificity.