Genome-wide detection of unknown subtle mutations in bacteria by combination of MutS and RDA

We propose a procedure for detecting unknown, subtle DNA changes throughout the entire bacterial genome by a combination of MutS and RDA. Current techniques detect subtle mutations after PCR amplification of the target regions, so the mutation detection is done between amplified PCR fragments. In this paper, genome-wide subtle mutation scanning in bacteria was performed by combining the MutS and RDA techniques. Our strategy for cloning a small mutation region is composed of two steps: an enrichment of fragments containing subtle mutations using MutS, followed by an RDA subtraction procedure for further enrichment. We successfully identified small mutations such as a four-base insertion, a two-base insertion, and transition mutations in bacteria.     

The major defect in Ashkenazi Jews with Tay-Sachs disease is an insertion in the gene for the alpha-chain of beta-hexosaminidase

The Ashkenazi Jewish population is enriched for carriers of a fatal form of Tay-Sachs disease, an inherited disorder caused by mutations in the alpha-chain of the lysosomal enzyme, beta-hexosaminidase A. Until recently it was presumed that Tay-Sachs patients from this ethnic isolate harbored the same alpha-chain mutation. This was disproved by identification of a splice junction defect in the alpha-chain of an Ashkenazi patient which could be found in only 20-30% of the Ashkenazi carriers tested. In this study we have isolated the alpha-chain gene from an Ashkenazi Jewish patient, GM515, with classic Tay-Sachs disease who was negative for the splice junction defect. Sequence analysis of the promoter region, exon and splice junctions regions, and polyadenylation signal area revealed a 4-base pair insertion in exon 11. This mutation introduces a premature termination signal in exon 11 which results in a deficiency of mRNA in Ashkenazi patients. A dot blot assay was developed to screen patients and heterozygote carriers for the insertion mutation. The lesion was found in approximately 70% of the carriers tested, thereby distinguishing it as the major defect underlying Tay-Sachs disease in the Ashkenazi Jewish population.     

APEX disease gene resequencing: mutations in exon 7 of the p53 tumor suppressor gene.

Detection of mutations in disease genes will be a significant application of genomic research. Methods for detecting mutations at the single nucleotide level are required in highly mutated genes such as the tumor suppressor p53. Resequencing of an individual patient's DNA by conventional Sanger methods is impractical, calling for novel methods for sequence analysis. Toward this end, an arrayed primer extension (APEX) method for identifying sequence alterations in primary DNA structure was developed. A two-dimensional array of immobilized primers (DNA chip) was fabricated to scan p53 exon 7 by single bases. Primers were immobilized with 200 microm spacing on a glass support. Oligonucleotide templates of length 72 were used to study individual APEX resequencing reactions. A template-dependent DNA polymerase extension was performed on the chip using fluorescein-labeled dideoxynucleotides (ddNTPs). Labeled primers were evanescently excited and the induced fluorescence was imaged by CCD. The average signal-to-noise ratio (S/N) observed was 30:1. Software was developed to analyze high-density DNA chips for sequence alterations. Deletion, insertion, and substitution mutations were detected. APEX can be used to scan for any mutation (up to two-base insertions) in a known region of DNA by fabricating a DNA chip comprising complementary primers addressing each nucleotide in the wild-type sequence. Since APEX is a parallel method for determining DNA sequence, the time required to assay a region is independent of its length. APEX has a high level of accuracy, is sequence-based, and can be miniaturized to analyze a large DNA region with minimal reagents.     

Potassium permanganate and tetraethylammonium chloride are a safe and effective substitute for osmium tetroxide in solid-phase fluorescent chemical cleavage of mismatch.

Whilst chemical cleavage of mismatch (CCM) detects all point mutations in DNA, its widespread use has been hampered by the complex multistage methodology and the need for toxic chemicals, in particular osmium tetroxide. Here we show that osmium tetroxide can be replaced by potassium permanganate, giving the same spectrum of mutation detection, but with greater sensitivity. The use of potassium permanganate is compatible with solid phase capture and fluorescent detection, giving a safer method of mutation detection. We present here a comparison of CCM with osmium tetroxide and with potassium permanganate, tested on a complete set of single base pair mismatches and a number of small insertion/deletions.     

Identification of a human specificAlu insertion in the factor XIIIB gene

The factor XIIIB gene was examined to determine the nature of a previously described 300 bp restriction fragment length polymorphism (RFLP) seen in the human population. Polymerase chain reaction analysis of different regions within the factor XIIIB gene was carried out to define a high resolution map of the region encompassing the polymorphism, followed by DNA sequence analysis. AnAlu insertion was found to be the source of this variation. ThisAlu repeat is a member of the human specific-1 (HS-1) subfamily, although one of the five diagnostic nucleotides is a cattarhine specific (CS) subfamily mutation, suggesting that it may represent an intermediate form in the evolution between these two subfamilies. Subsequently, we developed a PCR-based assay to detect the polymorphism, rendering it a more useful marker for genetic linkage studies and genome mapping. This insertion is also a valuable polymorphism for human population studies, as demonstrated by the large variations in allele frequencies seen in three population groups.     

Mutation detection, interpretation, and applications in the clinical laboratory setting

Mutation detection plays an increasingly significant role in clinical diagnostic testing, posing formidable challenges for laboratories. The expanding indications for clinical molecular testing and the nuances of interpreting test results are discussed. Methods for screening mutation detection platforms and monitoring assay reliability are presented, and results of platform comparisons are described. The potential for irrevocable medical interventions following a positive mutation analysis is highlighted to stress the imperative for accuracy in mutation detection and vigilance in the clinical arena.     

Development of real-time PCR using Minor Groove Binding probe to monitor the biological control agent Candida oleophila (strain O)

A real-time PCR assay using a 3'-Minor Groove Binding (MGB) probe was developed for specific detection and monitoring of Candida oleophila (strain O), a biocontrol agent against Botrytis cinerea and Penicillium expansum, on harvested apples. The application of the RAPD technique on C. oleophila strains followed by reproducible sequence characterized amplified region (SCAR) amplifications allowed the identification of a semi-specific fragment of 244 bp, observed in the profiles of strain O and three other C. oleophila strains. After sequencing, polymorphisms (3%) were observed between the strain O sequence and the three other sequences. A 3'-Minor Groove Binding probe was designed to specifically match a region of the strain O sequence and was able to discriminate a single base mutation or a two-base difference in the corresponding sequences of the non-target strains. This specific detection method was applied to monitor strain O population, recovered by a washing buffer, from harvested apples. Population densities were calculated using an external standard curve consisting in a serial dilution of strain O cells in the washing buffer from untreated apples. Linearity in the standard curve was kept between 1.64 x 10(2) and 1.64 x 10(5) cfu cm(-2) of apple surface. During a first practical experiment, the calculated population densities were similar to those obtained by plating on semi-selective media. This new real-time PCR method is a promising tool to monitor quickly and specifically strain O population on apple surface in middle- or large-scale experiments.     

Application of dHPLC for mutation detection of the fibrillin-1 gene for the diagnosis of Marfan syndrome in a National Health Service Laboratory

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder caused by mutations in the fibrillin-1 gene FBN1. Mutation detection of this 65-exon gene presents a particular challenge for the diagnostic service in cost, time constraints, and the need to maintain a stringently optimized assay procedure. Using denaturing high-performance liquid chromatography (dHPLC), we have designed a procedure for rapid mutation scanning, redesigning 50% of published primer sets, screening by Ensembl to avoid inclusion of polymorphic variations and employing a limited set of PCR conditions to allow for a high-throughput 96-well format. We have screened 262 unrelated patients with MFS or Marfan-like phenotypes and detected 103 (39.3%) mutations including 93 different mutations, 72 of which are novel. The mutations include 55 missense (53.4%) 19 splice site (18.5%), 17 frameshift (16.5%), 11 nonsense (10.7%) and 1 in-frame deletion/insertion.     

A dithio-coupled kinase and ATPase assay

Kinases and ATPases produce adenosine diphosphate (ADP) as a common product, so an assay that detects ADP would provide a universal means for activity-based screening of enzymes in these families. Because it is known that most kinases accept ATPbetaS (sulfur on the beta-phosphorous) as a substrate in place of adenosine triphosphate (ATP), the authors have developed a continuous assay using this substrate, with detection of the ADPbetaS product using dithio reagents. Such an assay is possible because dithio groups react selectively with ADPbetaS and not with ATPbetaS. Thiol detection was done using both Ellman's reagent (DTNB) and a recently developed fluorescent dithio reagent, DSSA. Therefore, the assay can be run in both absorbance and fluorescence detection modes. The assay was used to perform steady-state kinetic analyses of both hexokinase and myosin ATPase. It was also used to demonstrate the diastereoselectivity of hexokinase (R) and myosin ATPase (S) for the isomers of ATPbetaS, consistent with previous results. When run in fluorescence mode using a plate reader, an average Z' value of 0.54 was obtained, suggesting the assay is appropriate for high-throughput screening.     

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.     

Screening for mutations in human HPRT cDNA using the polymerase chain reaction (PCR) in combination with constant denaturant gel electrophoresis (CDGE).

Previously, we reported the modification of denaturing gradient gel electrophoresis called constant denaturant gel electrophoresis (CDGE). CDGE separates mutant fragments in specific melting domains. CDGE seems to be a useful tool in mutation detection. Since the hypoxanthine phosphoribosyltransferase (HPRT) gene is widely used as target locus for mutation studies in vitro and in vivo, we have examined the approach of analyzing human HPRT cDNA by polymerase chain reaction (PCR) and CDGE. All nine HPRT exons are included in a 716-bp cDNA fragment obtained by PCR using HPRT cDNA as template. When the full-length cDNA fragment was examined by CDGE, it was possible to detect mutations only in the last part of exon 8 and exon 9. However, digestion of the cDNA fragment with the restriction enzyme AvaI prior to CDGE enabled us to detect point mutations in most of exon 2, the beginning of exon 3, the last part of exon 8 and exon 9. With the use of two internal primer sets, including a GC-rich clamp on one of the primers in each pair, a region containing most of exon 3 through exon 6 was amplified and we were able to resolve fragments with point mutations in this region from wild-type DNA. The approach described here allows for rapid screening of point mutations in about two thirds of the human HPRT cDNA sequence. In a test of this approach, we were able to resolve 12 of 13 known mutants. The mutant panel included one single-base deletion, one two-base deletion and 11 single-base substitutions.     

A method to quantitatively detect H-ras point mutation based on electrochemiluminescence

Conventional methods for point mutation detection are usually multi-stage, laborious, and need to use radioactive isotopes or other hazardous materials, and the assay results are often semi-quantitative. In this work, a protocol for quantitative detection of H-ras point mutation was developed. Electrochemiluminescence (ECL) assay was coupled with restriction endonuclease digestion directly from PCR products. Only the wild-type amplicon containing the endonuclease's recognition site can be cut off, and thus cannot be detected by ECL assay. Using the PCR-ECL method, 30 bladder cancer samples were analyzed for possible point mutation at codon 12 of H-ras oncogene. The results show that the detection limit for H-ras amplicon is 100 fmol and the linear range is more than three orders of magnitude. The point mutation was found in 14 (46.7%) out of 30 bladder cancer samples. The experiment results demonstrate that the PCR-ECL method is a feasible quantitative approach for point mutation detection due to its safety, high sensitivity, and simplicity.     

A very fast ion-pair reversed-phase HPLC method for the separation of the most significant nucleotides and their degradation products in human red blood cells

A simple and fast ion pair reversed-phase high-performance liquid chromatographic method has been developed for the simultaneous determination of ATP, ADP, AMP, GTP, GDP, IMP, NADP+, NADPH+, NAD+, NADH, ADP-ribose, inosine, adenosine, hypoxanthine, and xanthine. This method allows us to have a complete picture of the most important nucleotides present in fresh human erythrocytes. Furthermore it is particularly useful in the study of the erythrocyte adenine nucleotide catabolism allowing the detection of degradation products such as IMP, inosine, adenosine, hypoxanthine, and xanthine. The separation of the compounds under investigation is achieved in less than 15 min using a reversed-phase 3-micron Supelcosil LC-18 column and adding tetrabutylammonium, as ion-pair agent, to the buffers. The short time of analysis, the high reproducibility of the system, and the accurate evaluation of the compounds of interest make this method particularly suitable for routine analysis. Finally it is possible to use this assay as an alternative method of measuring activities of enzymes which catalyze reactions involving some of these compounds, as in the case of Na+-K+ ATPase, AMP deaminase, and adenosine deaminase.     

电化学发光PCR方法检测结肠癌中K-ras癌基因点突变

发展了一种可用于快速检测K-ras癌基因点突变的电化学发光PCR(ECL-PCR)分析方法,该法采用三联吡啶钌标记的上游引物和生物素标记的下游引物对目的片段进行PCR扩增;随后,采用限制性内切酶MvaI对扩增产物进行酶切,由于突变导致酶切位点的丢失,所以只有野生型样品能被切断;通过生物素与链霉亲和素包被的磁珠连接,将生物素标记的DNA片段收集到反应池中进行电化学发光检测。采用该法对20例结肠癌组织中的K-ras癌基因第12位密码子进行点突变分析,得出其中有9例存在点突变,点突变率为45%。该方法操作简便、安全、快速、灵敏,可用于快速检测K-ras癌基因点突变。     

Mechanism of frameshift (deletion) generated by acetylaminofluorene-derived DNA adducts in vitro

We have investigated the mechanism of frameshift (deletion) mutagenesis induced by acetylaminofluorene- (AAF-) derived DNA adducts. dG-AAF-modified oligodeoxynucleotides, with different bases positioned 5' to the lesion, were annealed to (32)P-labeled 13-mer primers and then used in primer extension reactions catalyzed by the 3'-->5' exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I. When the dNMP positioned opposite dG-AAF could pair with its complementary base at the 5' flanking position, single-base deletions were produced at high frequency. Similarly, when the complementary base was two positions 5' to the dG-AAF, two-base deletions occurred. The relative frequency of base insertions opposite dG-AAF followed the order dCMP > dAMP > dGMP > dTMP; the frequency of dNTP insertion opposite the lesion paralleled the formation of frameshift deletions. When a template designed to induce three-base deletions was used for translesion synthesis catalyzed by the exo(-) Klenow fragment, the expected three-base deletion was formed. When dG-AAF-modified templates containing iterated bases 5' to the lesion were annealed to primers with the complementary dNMP positioned opposite the lesion, the dNMP inserted opposite the dG-AAF tended to pair with the complementary base 5' to the lesion, thereby forming shorter deletions. Taken together, these results support the molecular mechanism for frameshift deletion proposed earlier by Shibutani and Grollman in which direct base insertion precedes misalignment [(1993) J. Biol. Chem. 268, 11703].     

Two-base DNA hairpin-loop structures in vivo.

In vitro studies have revealed that DNA hairpin-loops usually contain four unpaired bases. However, a small subset of sequences can form two-base loops. We have previously described an in vivo assay that is sensitive to tight loop formation and have set out to test whether DNA sequences known to form two-base loops in vitro also form tight loops in vivo. It is shown that the sequences 5'dCNNG and 5'dTNNA behave as predicted if they favour two-base loop formation in vivo, a result that is consistent with previously described in vitro studies. The ability of specific DNA sequences to form tight loops in vivo has implications for their potential to form transient structures involved in gene regulation, recombination and mutagenesis.     

Determination of the rate of base-pair substitution and insertion mutations in retrovirus replication.

We recently described a protocol for determination of retrovirus mutation rates, that is, the mutation frequency in a single cycle of retrovirus replication (J.P. Dougherty and H.M. Temin, Mol. Cell. Biol. 6:4378-4395, 1987; J.P. Dougherty and H.M. Temin, p. 18-23, in J. H. Miller and M. P. Calos, ed., Gene Transfer Vectors for Mammalian Cells, 1987). We used this protocol to determine the mutation rates for defined mutations in a replicating retrovirus by using a spleen necrosis virus-based vector. We determined that the mutation rate for a single base pair substitution during replication of this avian retrovirus is 2 x 10(-5) per base pair per replication cycle and the insertion rate is 10(-7) per base pair per replication cycle. It will be possible to use this protocol to determine mutation rates for other retroviruses.     

Double-strand cleavage at a two-base deletion mismatch in a DNA heteroduplex by nuclease S1

A two-base deletion mismatch was generated in a DNA heteroduplex by hybridization of two linear plasmid DNA molecules differing only by the presence of a two-base deletion in one of them. The heteroduplex was shown to be sensitive to double-strand cleavage by nuclease S1, thus demonstrating the potential value of single-stranded probes for the detection of polymorphisms in genomic DNA due to very small deletions.     

ETA基因作为荧光定量PCR靶基因设计TaqMan探针快速检测铜绿假单胞菌的研究

铜绿假单胞菌是临床上常见致病菌, 传统的检测方法有各种弊端。本研究对该细菌的ETA基因用生物信息学方法加以分析, 选取相对保守且高度特异的DNA序列, 设计一对特异性引物和一个TaqMan探针, 建立FQ-PCR (fluorescence quantitative PCR)检测PA的方法。通过对梯度浓度的铜绿假单胞菌基因组DNA样品进行FQ-PCR检测和对多种细菌的DNA进行扩增, 来检测其灵敏度和验证引物和探针的特异性。试验结果表明, 对比现有的检测方法, 以ETA基因为靶基因, 基于TaqMan探针的快速FQ-PCR检测技术有更高的灵敏度和更好的特异性等优点, 具有很好的研究价值和应用前景。     

ETA基因作为荧光定量PCR靶基因设计TaqMan探针快速检测铜绿假单胞菌的研究

铜绿假单胞菌是临床上常见致病菌, 传统的检测方法有各种弊端。本研究对该细菌的ETA基因用生物信息学方法加以分析, 选取相对保守且高度特异的DNA序列, 设计一对特异性引物和一个TaqMan探针, 建立FQ-PCR (fluorescence quantitative PCR)检测PA的方法。通过对梯度浓度的铜绿假单胞菌基因组DNA样品进行FQ-PCR检测和对多种细菌的DNA进行扩增, 来检测其灵敏度和验证引物和探针的特异性。试验结果表明, 对比现有的检测方法, 以ETA基因为靶基因, 基于TaqMan探针的快速FQ-PCR检测技术有更高的灵敏度和更好的特异性等优点, 具有很好的研究价值和应用前景。