Detection of virtually all mutations-SSCP (DOVAM-S): a rapid method for mutation scanning with virtually 100% sensitivity.

Dideoxy fingerprinting (ddF) was used as a tool to search for a generic set of conditions with sufficient power to detect virtually all mutations. For each condition tested, a very large sample of mutation-containing, single-stranded segments (about 1500) were analyzed with ddF. Correlation coefficients identified pairs of conditions in which single-strand conformation polymorphism (SSCP) mobilities were poorly correlated. The data strongly suggest that tertiary structure (e.g., base-sugar and sugar-sugar interactions) rather than secondary structure is the predominant determinant of mobility shifts by SSCP. Five conditions were selected with sufficient redundancy to detect all the mutations. The sensitivity of detection of virtually all mutations-SSCP (DOVAM-S) was determined by blinded analyses on samples containing additional mutations scattered throughout the eight exons and splice junctions in the factor IX gene. The factor IX gene sequence (2.5 kb) was scanned in one lane by 15 PCR-amplified segments (125 kb of sequence scanned per gel). All of the 84 single-base substitutions were detected in the blinded analyses, the first consisting of 50 hemizygous mutant and wild-type (WT) samples and the second consisting of 50 heterozygous mutant and WT samples. DOVAM-S is estimated to be five times faster than fluorescent DNA sequencing for the detection of virtually all mutations when the five conditions are applied.     

Carrier and prenatal diagnosis of X-linked severe combined immunodeficiency: mutation detection methods and utilization

IL2RG, the gene encoding the common γ chain, γc, of the receptor for interleukin-2 and other cytokines, has been identified as the disease gene for severe combined immunodeficiency (SCID) of the X-linked type. Specific mutational diagnosis for X-linked SCID has thus become possible. For many women at risk for carrying an IL2RG mutation, no samples were saved from an affected male relative prior to either death or bone marrow transplantation (BMT). To establish optimal methods for genetic evaluation of such women, we compared mutational screening by single-strand conformational polymorphism, heteroduplex analysis and dideoxy fingerprinting (ddF). Abnormally migrating band patterns were followed up with direct sequencing for identification of specific mutations. The most sensitive method, ddF, detected heterozygous alterations, subsequently confirmed to represent significant mutations, in all of 19 unrelated obligate or suspected carriers studied. Some of these women, as well as others at risk for carrying an X-linked SCID mutation, enrolled in a study of prenatal diagnosis after fetal testing for gender determination. Originally using linkage analysis and, more recently, specific detection of IL2RG mutations, we evaluated pregnancies at risk for X-linked SCID prospectively on a research basis. Of 27 male fetuses tested 14 were predicted to be unaffected and confirmed to have normal immune status at birth. Among pregnancies predicted to be affected, 2 were terminated, while 11 affected males were born at term. Nine of these received neonatal BMT, one had BMT at 3 months of age, and one underwent a successful experimental in utero BMT. In our study cohort accurate prenatal diagnosis assisted decision making and expanded treatment options for families at risk for having infants with a severe, but treatable genetic disorder that presents early in life. Received: 4 October 1996     

Fluorescence-based directed termination PCR: direct mutation characterization without sequencing

We describe a fluorescence-based directed termination PCR (fluorescent DT–PCR) that allows accurate determination of actual sequence changes without dideoxy DNA sequencing. This is achieved using near infrared dye-labeled primers and performing two PCR reactions under low and unbalanced dNTP concentrations. Visualization of resulting termination fragments is accomplished with a dual dye Li-cor DNA sequencer. As each DT–PCR reaction generates two sets of terminating fragments, a pair of complementary reactions with limiting dATP and dCTP collectively provide information on the entire sequence of a target DNA, allowing an accurate determination of any base change. Blind analysis of 78 mutants of the supF reporter gene using fluorescent DT–PCR not only correctly determined the nature and position of all types of substitution mutations in the supF gene, but also allowed rapid scanning of the signature sequences among identical mutations. The method provides simplicity in the generation of terminating fragments and 100% accuracy in mutation characterization. Fluorescent DT–PCR was successfully used to generate a UV-induced spectrum of mutations in the supF gene following replication on a single plate of human DNA repair-deficient cells. We anticipate that the automated DT–PCR method will serve as a cost-effective alternative to dideoxy sequencing in studies involving large-scale analysis for nucleotide sequence changes.     

结核分枝杆菌rpoB基因突变的检测(简报)

结核病主要是由结核分枝杆菌(Mycobacterium tuberculosis)引起的一种慢性传染性疾病。利福平是结核病化疗方案中一个关键性的药物,它在结核病的短程化疗中起着重要的作用。但是,在我国结核菌对利福平的耐药发生率呈上升局势,而通过传统的依赖生物生长的药敏试验方法进行结核菌对利福平耐药性检测所需时间较长(4-8周),不能满足临床早期开展有效化疗的需要,所以迫切需要建     

Diversity of the archaeal community in 44 anaerobic digesters as determined by single strand conformation polymorphism analysis and 16S rDNA sequencing

The diversity of Archaea in anaerobic digesters was characterized by strand conformation polymorphism (SSCP) analysis and the sequencing of 16S rDNA genes. The 44 digesters sampled, located in eight different countries, treated effluents from agriculture, the food processing and petro-chemical industries, pulp and paper plant, breweries, slaughterhouses and municipal waste. All the existing processes were represented among the samples (fixed-film, fluidized bed, stirred-tank, UASB, sequential batch reactor, lagoon). Single strand conformation polymorphism analysis targeting the V3 region of 16S rDNA revealed between four to six distinct archaeal peaks per digester. The diversity of dominant Archaea in the 44 digesters was estimated as 23 different 16S rDNA sequences. Cloning of archaeal 16S rRNA genes from 11 distinct total genomic DNA, screening of clones by SSCP and the sequencing of 170 of them made it possible to characterize these SSCP peaks. All the sequences retrieved were members of the Euryarchaeaota subdomain. Furthermore, most of the sequences retrieved were very close to already known and cultivated strains or to environmental clones. The most frequent archaeal sequences were close to Methanosaeta concilii and to a 16S rDNA clone vadinDC06 located in the Methanobacterium clade (84% and 73% of digesters respectively). The other sequences were members of the Methanobacteriales and the Methanomicrobiales families. Only one sequence was far from any sequence of the database and it could be grouped with several sequences of environmental clones. Each digester harboured between two to nine archaeal sequences with only one of them corresponding to a putative acetate-utilizing species. Furthermore, the process in the digesters appeared to play a part in the distribution of archaeal diversity.     

Single-stranded conformation polymorphism (SSCP)-driven indirect sequencing in detection of short deletion

To seek for novel rare and/or sporadic mutations within genomic neighborhood of common −344 C>T (rs2427827) FCER1A proximal promoter polymorphism, which by its prevalence could have masked the presence of less frequent genetic variants in our previous single-stranded conformational polymorphism (SSCP) mutational search study, SSCP screening was repeated using primers fixing −344 C>T variant. The genomic region surrounding −344 C>T polymorphism was confirmed to be fairly conservative and only a single sporadic mutation (present in 1 out of 196 chromosomes), a 6-bp deletion −262 to 257 del CTAGAC, was found. From the methodological point of view, we demonstrated a successful detection of a short-length polymorphism using SSCP screening in a population, in which the same genomic region contained frequent single-nucleotide polymorphic variant. In conjunction with subsequent cloning of aberrantly migrating PCR products and SSCP-driven indirect sequencing of the clones, this method offers a superior (to direct sequencing of PCR products) detection of rare mutations. The cost-effective method applied by us enables a reliable characterization of infrequent (thus present only in heterozygotic form) short-length variance of the sequence which are difficult to interpret by direct sequencing.     

Mutational analysis by a combined application of the multiple restriction fragment-single strand conformation polymorphism and the direct linear amplification DNA sequencing protocols.

We describe here an improved procedure for polymerase chain reaction (PCR)-based single strand conformation polymorphism (SSCP) for rapid mutational detection. To circumvent the restriction of having to analyze relatively short PCR fragments, restriction endonucleases were used to cleave a longer PCR product and the mixture of fragments was analyzed directly in SSCP gel electrophoresis. This multiple restriction fragment (MRF)-SSCP protocol was demonstrated by the detection of a 4-bp deletion in codons 41-42 and a point mutation in the IVS-2 sequence of the human beta-globin gene. The MRF-SSCP or the standard SSCP protocol was then combined with the linear amplification DNA sequencing (LADS) procedure for direct analysis of the PCR products without further purification for an exact characterization of the mutations detected. In the LADS analysis, homo- or heterozygosity of a mutation was easily distinguished by the appearance of a single- or double-lane band in the sequencing gel. The choice of isotope used and different labeling methods were compared and were found, in some cases, to produce SSCP patterns of different complexities. The combined MRF-SSCP/LADS protocol permits rapid mutational analysis of a large number of clinical samples using only very small amounts of materials and can easily be adopted for nonisotopic clinical applications.     

A comparison of BRCA1 mutation analysis by direct sequencing, SSCP and DHPLC

The most sensitive screening technique for genes that predispose patients for particular cancers is direct sequencing. However, sequencing of complex genes is technically demanding, costly and time-consuming. We have tested alternate screening techniques to find a fast sensitive method for detecting alterations of DNA in the large BRCA1 gene prior to sequencing. Sequencing of this gene is particularly arduous because it lacks clearly defined mutation sites. The single-strand conformation polymorphism (SSCP) technique is one of the most frequently used pre-screening methods but its sensitivity and efficiency is not completely satisfying. We have compared the SSCP assay with a newly developed technique called denaturing high performance liquid chromatography (DHPLC) to screen the BRCA1gene. We studied 23 patients at high risk for early onset breast and ovarian cancer and four controls. In these patients, a total of 113 fragments with sequence variations in the BRCA1 gene could be identified. The DHPLC technique resolved 100% of the DNA alterations that were observed in cycle sequencing. In contrast, mutation analysis by SSCP accounted for 94% of the detected variations. In addition, DHPLC screening allowed us to discriminate between different alterations in a single fragment, because of the characteristic elution profiles of the DNA molecules. Polymorphisms that were present in our samples could be predicted by means of DHPLC testing independently of sequence analysis. We conclude that DHPLC is a highly potent screening method for genetic analyses. It is highly sensitive, efficient and economical and can be automated. Electronic Publication     

Use of transposon-promoted deletions in DNA sequence analysis

The usefulness of the dideoxy method for DNA sequencing can be greatly extended by the use of transposon-generated deletions. These deletions have the unique property of extending from a fixed nucleotide at the transposon terminus to various sites outside it. A plasmid (pAA3.7) carrying Tn9, which allows positive selection of such deletions as galactose-resistant colonies of Escherichia coli, is described. A cloned gene can thus be subdivided into a series of overlapping sequences, all of which are fused to a common sequence at the transposon terminus. Restriction fragments carrying the segments fused by deletions are cloned in M13, and sequenced using a primer complementary to the Tn9 terminus. Complete nucleotide sequence of the gene is assembled from sequence overlaps found in deletions with end-points approximately 350 base-pairs apart. The method is rapid, requires minimal in vitro manipulation, and is free from redundant information normally produced in shotgun sequencing.     

Sequencing of large double-stranded DNA using the dideoxy sequencing technique

The dideoxy sequencing technique has been applied to the direct sequencing of large double-stranded DNA molecules with a small single-stranded primer. For instance, the method was applied to the lambda genome, which contains 48 502 base-pairs (Sanger F, Coulson AR, Hong GF, Hill D & Petersen GB, 1982, J. Mol. Biol., in press), and the coding region for gene W identified. The procedure proves useful in the sequence analysis of a large number of different mutations in a particular region and in the analysis of eukaryotic DNA cloned in plasmids, phages, and cosmids.     

SNPs of hemocyanin C-terminal fragment in shrimp Litopenaeus vannamei

In this study, we identified a variable region in the C-terminus of hemocyanin from the shrimp Litopenaeus vannamei (2288-2503bp, HcSC) by sequence alignments. A total of 13 SNPs were identified by PCR-SSCP and HcSC clone sequencing. The SSCP patterns of HcSC could be modulated in Vibro parahaemolyticus-treated shrimps. A novel SSCP band with four SNP sites was identified in V. parahaemolyticus-resistant shrimps. More importantly, three of these four SNPs introduced variations in amino acid sequence and possibly secondary structure of the HcSC polypeptide and resulted in a higher agglutinative activity against seven pathogenic bacteria. These results suggest that the C-terminus of shrimp L. vannamei hemocyanin possesses SNPs, which may be related to shrimp resistance to different pathogens.     

Scanning by DOVAM-S detects all unique sequence changes in blinded analyses: evidence that the scanning conditions are generic

The [detection of virtually all mutations]-SSCP (DOVAM-S) is a highly sensitive variant of single strand conformation polymorphism (SSCP). Mutations in the factor IX gene were used to find a set of five SSCP conditions that detects virtually all mutations. A blinded analysis of the factor IX gene in patients with hemophilia B detected 82 of 82 unique mutations. Since the method was developed and tested on the factor IX gene, it is possible that the conditions selected work more efficiently in the factor IX gene than in other genes. To test the general applicability of the conditions under which DOVAM-S detected all mutations in this gene, blinded analyses were performed in the human factor VIII and ataxia-telangiectasia (ATM) genes. Segments were amplified individually, combined into groups of 16 to 18 amplified segments and electrophoresed in five different nondenaturing conditions of varying matrices, buffers, temperatures and additives. Blinded analyses were performed in 92 samples from patients with hemophilia A (factor VIII gene) and 19 samples from A-T patients (ATM gene). Combined with an earlier blinded analysis in the factor IX gene, all of the 250 mutations and polymorphisms (180 of which are unique) were detected in both analyses. For two, three and four joint conditions, the average detection frequency ranged from 77%-97%, 91%-100% and 95%-100%, respectively. For each of the genes, one mutation may have been missed if only four conditions were used. With DOVAM-S, approximately 500 kb of autosomal sequence can be scanned in five gels with virtually 100% detection of mutations within the scanned region. The detection of 180 out of 180 unique sequence changes implies that DOVAM-S detects at least 96.5% (P = 0.03) of mutations. Blinded analyses that detect 400 unique sequence changes are required to determine that a scanning method detects at least 98.5% of mutations.     

Candidate gene analyses by scanning or brute force fluorescent sequencing: a comparison of DOVAM-S with gel-based and capillary-based sequencing

For epidemiological and diagnostic applications, detection of virtually all mutations is desired. Herein, blinded analyses of DOVAM-S (Detection Of Virtually All Mutations-SSCP), a robotically enhanced multiplex SSCP method, demonstrate that all of 525 mutations (391 unique) are detected by the method. In addition, the costs of DOVAM-S, gel-based fluorescent sequencing and capillary-based fluorescent sequencing are compared. The relative cost effectiveness of gel-based and capillary-based sequence analysis depends on throughput and whether depreciation and service are considered. DOVAM-S reduces the cost of candidate gene analyses relative to brute force sequencing by about threefold.     

应用4种方法检测结核分枝杆菌临床分离株对链霉素的耐受性

通过DNA测序、SSCP、RFLP和反向斑点杂交技术分析167株结核分枝杆菌临床分离株的耐药基因型,评价结核分枝杆菌rpsL或rrs基因突变与链霉素（SM）耐受性之间的关系,比较4种分子方法检测SM耐受性的临床价值。98株耐SM分离株中,78株（79．6％）rpsL 43位或88位密码子错义突变导致赖氨酸置换为精氨酸,6株（6．1％）rrs 513位碱基A突变为C或T或516位C突变为T,14株（14．3％）未发现突变;69株SM敏感的分离株未发现这两个基因突变。应用SSCP、RFLP和RDBH方法分析上述突变和野生序列的结果与DNA测序完全一致,RDBH方法可从98株耐SM分离株中正确鉴定出84株（85．7％）分离株的5种突变基因型。结果表明,应用分子技术分析rpsL和rrs基因突变可快速检测大多数结核分枝杆菌对SM的耐受性,反向斑点杂交方法是一个快速、简便和可靠地检测药物耐受性的分子方法。     

Sequence alterations can mask each other's presence during screening with SSCP or heteroduplex analysis: BRCA genes as examples

For mutation detection, various screening techniques are widely used because DNA sequencing, the gold-standard method, is still considered to be expensive and laborious for high-throughput screening. Single-strand conformation polymorphism (SSCP) analysis, heteroduplex analysis (HA) and their variant techniques are popular and frequently used for this purpose. It is widely accepted that when searching for unknown sequence variations, any revealed distinct pattern should always be sequenced. We give examples here of the BRCA1 and BRCA2 genes where the SSCP/HA techniques can produce ambiguous predictions if used to detect known genetic variants compared to positive controls. Using direct DNA sequencing, we provide evidence that in such cases, mutations or polymorphisms can mask each other's presence. This phenomenon can often influence the results of any DNA testing because genetic variations such as single-nucleotide polymorphisms occur frequently in the human genome. We suggest that even in the case of known electrophoretic patterns of well-characterized genetic alterations, every sequence alteration should be confirmed by direct DNA sequencing, especially if genetic testing is carried out for diagnostic purposes.     

Imbalance of G and C contents influences the sensitivity of single-strand conformation polymorphism

After analyzing chicken ATPase6 by single-strand conformation polymorphism (SSCP), we observed two band species. But based on the sequencing results, we found two mutations, and one of them was not detected by SSCP. The primer P2 was designed to detect the additional mutation site, which was not detected by the original primer. And the P2 SSCP results then agreed with the sequencing data. Analysis of the percent of G and C bases and secondary structures indicated that it might be caused by a similar secondary structure which might be resulting from serious imbalance of G and C contents.     

Analysis of gyrA mutations in quinolone-resistant and -susceptible Campylobacter jejuni isolates from retail poultry and human clinical isolates by non-radioactive single-strand conformation polymorphism analysis and DNA sequencing

AIMS: The aims of this study were to characterize the molecular variations in the quinolone resistance-determining region (QRDR) of gyrA among quinolone-resistant and -susceptible Campylobacter jejuni isolates originating from foods of animal origin and human infections and to evaluate the suitability of the single-strand conformation polymorphism (SSCP) method as a screening method for molecular characterization of fluoroquinolone resistance. METHODS AND RESULTS: Alterations in QRDR of gyrA from 182 C. jejuni isolates were determined by nonradioisotopic SSCP analysis and direct sequencing. A total of 13 types of nucleic acid sequence combinations within the QRDR of the gyrA gene resulted in 11 different SSCP patterns. All nalidixic acid resistant strains possessed nucleotide substitution at either codon Thr-86 or Asp-90. Silent mutations were detected additionally. Thr-86 to Ile mutation was detected in all 139 ciprofloxacin resistant strains, which showed cross-resistance to nalidixic acid. CONCLUSIONS: The SSCP method is suitable for a molecular screening of quinolone resistant C. jejuni isolates and in combination with DNA sequencing suitable to detect genetic variations of the QRDR of gyrA. SIGNIFICANCE AND IMPACT OF STUDY: This study provides data of the genetic variations of the QRDR of gyrA from C. jejuni isolates of foods and human beings.     

A vector for sequencing long (40-kb) DNA fragments

An improved vector (pAA113M) has been constructed for sequencing long (40-kb) DNA fragments. The DNA fragment is cloned in the tet gene of the cosmid and subdivided into numerous overlapping segments by IS1-promoted deletions. Plasmids bearing these deletions are fractionated by gel electrophoresis, and shortened further from the opposite end by treatment with specific restriction endonucleases. Thus, a series of short overlapping segments, spread across the entire length of the fragment, become fused to IS1. Each segment can then be sequenced by the dideoxy method using an IS1 primer. The plasmids can replicate either from their normal origin or, in the presence of a filamentous helper phage, from the M13 origin. Hence, each segment can be sequenced as either double-stranded DNA or single-stranded DNA. Sequences of IS1-promoted and restriction enzyme-generated deletions contain overlaps that can be used to assemble the complete 40-kb sequence.