Chemical Cleavage Of Mismatch Research Articles

Insight into an Oxidative DNA-Cleaving DNAzyme: Multiple Cofactors, the Catalytic Core Map and a Highly Efficient Variant.

SummaryAn oxidative DNA-cleaving DNAzyme (PL) employs a double-cofactor model “X/Cu2+” for catalysis. Herein, we verified that reduced nicotinamide adenine dinucleotide (NADH), flavin mononucleotide, cysteine, dithiothreitol, catechol, resorcinol, hydroquinone, phloroglucinol, o-phenylenediamine, 3,3′,5,5'-tetramethylbenzidine, and hydroxylamine acted as cofactor X. According to their structural similarities or fluorescence property, we further confirmed that reduced nicotinamide adenine dinucleotide phosphate (NADPH), 2-mercaptoethanol, dopamine, chlorogenic acid, resveratrol, and 5-carboxyfluorescein also functioned as cofactor X. Superoxide anions might be the commonality behind these cofactors. We subsequently determined the conservative change of individual nucleotides in the catalytic core under four different cofactor X. The nucleotides A4 and C5 are highly conserved, whereas the conservative levels of other nucleotides are dependent on the types of cofactor X. Moreover, we observed that the minor change in the PL's secondary structure affects electrophoretic mobility. Finally, we characterized a highly efficient variant T3G and converted its double-cofactor NADH/Cu2+ to sole-cofactor NADH.

Capture of somatic mtDNA point mutations with severe effects on oxidative phosphorylation in synaptosome cybrid clones from human brain.

Mitochondrial DNA (mtDNA) is replicated throughout life in postmitotic cells, resulting in higher levels of somatic mutation than in nuclear genes. However, controversy remains as to the importance of low-level mtDNA somatic mutants in cancerous and normal human tissues. To capture somatic mtDNA mutations for functional analysis, we generated synaptosome cybrids from synaptic endings isolated from fresh hippocampus and cortex brain biopsies. We analyzed the whole mtDNA genome from 120 cybrid clones derived from four individual donors by chemical cleavage of mismatch and Sanger sequencing, scanning around two million base pairs. Seventeen different somatic point mutations were identified, including eight coding region mutations, four of which result in frameshifts. Examination of one cybrid clone with a novel m.2949_2953delCTATT mutation in MT-RNR2 (which encodes mitochondrial 16S rRNA) revealed a severe disruption of mtDNA-encoded protein translation. We also performed functional studies on a homoplasmic nonsense mutation in MT-ND1, previously reported in oncocytomas, and show that both ATP generation and the stability of oxidative phosphorylation complex I are disrupted. As the mtDNA remains locked against direct genetic manipulation, we demonstrate that the synaptosome cybrid approach can capture biologically relevant mtDNA mutants in vitro to study effects on mitochondrial respiratory chain function.

P.P2063S: a neutral VWF variant masquerading as a mutation

Dear Editor, Kasatkar and colleagues recently reported that the previously described von Willebrand factor (VWF) variant c.6187C>T (p.P2063S) appears to be a common cause of type 3 von Willebrand disease (VWD3) in the Indian population [1]. In support of their conclusion, p.P2063S was the only VWF variant observed in 11 Indian VWD3 index cases (IC), tracked with disease phenotype and was predicted to be deleterious via in silico protein prediction analysis. In order to ensure accurate molecular characterisation of VWF variants, it is essential to consider all available information. Regarding p.P2063S, there are several lines of evidence that contradict the conclusion drawn. p.P2063S is also observed in healthy controls with reported population frequencies between 0.6–2.5% in African-Americans / Caucasians [2–4] and (as reported by Kasatkar and colleagues [1]) 3% in Indians. Given a frequency of 3% in the general population, Hardy-Weinberg equilibrium predicts homozygosity for this variant in approximately 1/1100 individuals. This would suggest a high population frequency of VWD3 if this variant were causative. It is also notable that p.P2063S has not been reported as causal in previous studies of VWD3 in Indians [5–7]. The majority of missense mutations reported in VWD3 patients occur in the VWF propeptide and C-terminus where they are reported to affect protein folding and multimerisation resulting in intracellular retention of the protein [2,7,8]. Kasatkar and colleagues argue that diagnosis of VWD3 relies on defective release or synthesis of VWF [1], however given that p.P2063S has no effect on VWF expression in vitro [2] it seems unlikely that this variant would affect VWF in vivo. This is supported by observations of p.P2063S in VWD patients inherited together with other VWF variants that more readily explain their VWD phenotype (Table 1), most notably in a Turkish VWD3 IC [8]. Table 1 Co-inheritance of p.P2063S and actual VWD causing variants In previous Indian VWD3 studies [5–7], molecular analysis failed to fully account for the genetic cause in 8/54 IC (15%). It is therefore likely that the actual disease causing mutation has yet to be identified by Kasatkar and colleagues, who imply that DNA scanning methods were used to identify mutations in their patient cohort [1]. DNA scanning methods are not 100% sensitive to all sequence variations so may fail to detect the ‘true’ mutation, for example chemical cleavage mismatch analysis identified a homozygous p.P2063S variant in a Turkish VWD3 IC but failed to identify the actual p.Q1734* causative mutation (Table 1) [8]. Alternatively, the causative mutation may have been overlooked if, for example, it were a synonymous variant that disrupts normal splicing or protein folding, or a deep intronic variant causing disruption of splicing outside the VWF regions analysed [9]. In summary, review of the available information supports the conclusion that p.P2063S is a common neutral VWF polymorphic variant. Further studies (for example using mRNA as the template investigated) may identify the underlying cause of VWD in this interesting patient group.

403 POSTER Specificity and sensitivity of point mutation detection in tumor suppressor genes via chemical cleavage of mismatches

403 POSTER Specificity and sensitivity of point mutation detection in tumor suppressor genes via chemical cleavage of mismatches

Chemical cleavage of DNA duplexes with single base mismatches as a basis for detection of random point mutations

The most promising approaches to detection of random point mutations are based on chemical cleavage of mismatches and other noncomplementarities. To demonstrate the specificity of this method, a model system was obtained for the first time as sets of 50-mer imperfect DNA duplexes containg all variants of mismatched and unpaired internal residues located in an invariant context and flanked by either A · T or G · C base pairs. Chemical cleavage of DNA duplexes immobilized on magnetic beads via the biotin-streptavidin interaction was accomplished using potassium permanganate or hydroxylamine, which are sensitive to the secondary DNA structure and react with thymine and cytosine, respectively. The reactivity of different mismatches was connected with the local duplex structure and depended on their type, orientation, and flanking nucleotides. The use of potassium permanganate and hydroxylamine to modify a heteroduplex mixture makes it possible to unambiguously detect a mismatch and, based on the type of reagent and the size of the cleavage products, to suppose the type and position of the mismatch and the flanking nucleotides. The model system can be used to evaluate the sensitivity of a chemical cleavage method and to control false-positive and false-negative results when different protocols are applied to the detection of DNA point mutations.

Experimental design in intraspecific organelle DNA sequence studies II: Haplotype detection by Chemical Cleavage of Mismatch (CCM) using SYBR® Green II staining

Studies of organelle DNA sequence at low taxonomic levels present a researcher with specific experimental design challenges. Such studies require sampling numerous individuals, many of which share identical haplotypes. The process is often limited by the time and money required for DNA sequencing. A number of screening techniques have been devised to combat this limitation, but many have not found wide‐spread use in botany. An ideal screening technique would be fast, easy, safe, inexpensive, detect 100% of mutations, be suitable for DNA fragments of about 1 kb, and not require complex equipment. This paper compares the heteroduplex analysis (HA) and chemical cleavage of mismatch (CCM) methods for detecting known point mutations and a deletion in a 1 kb region of non‐coding cpDNA from the Poa pratensis agg. A new CCM visualization method, staining with SYBR® Green II, was tested.

Chemical cleavage of mismatch (CCM) to locate base mismatches in heteroduplex DNA

This protocol describes the use of the chemical cleavage of mismatch (CCM) method to assess whether a region of DNA contains mutations and to localize them. Compared with other mutation-detection techniques (such as single strand-conformation polymorphism (SSCP) analysis, denaturing high-performance liquid chromatography (DHPLC) and denaturing gradient gel electrophoresis (DGGE)) that detect mutations in short DNA fragments and require highly specific melting temperatures, CCM has a higher diagnostic sensitivity suited to the detection of mutations in tumor genes, and can analyze amplicons < or = 2 kb in length. To detect mutations, PCR heteroduplexes are incubated with two mismatch-specific reagents. Hydroxylamine modifies unpaired cytosine and potassium permanganate modifies unpaired thymine. The samples are then incubated with piperidine, which cleaves the DNA backbone at the site of the modified mismatched base. Cleavage products are separated by electrophoresis, revealing the identity and location of the mutation. The CCM method can efficiently detect point mutations as well as insertions and deletions. This protocol can be completed in 10 h.

Mutation Detection by Solid-Phase Chemical Cleavage of Mismatches Using Radioactive Probes

This protocol is the first of two describing solid-phase chemical cleavage of mismatch (SpCCM), a method for the detection of mutations in genomic DNA. DNA fragments up to 2 kb in length can be analyzed to determine the position (within 10-15 bp) and identity of the mismatched nucleotide. While this protocol uses radioactively labeled probes and conventional gel electrophoresis followed by autoradiography, the second protocol employs fluorescently labeled primers and depends on an automated electrophoresis system with fluorescence detection Mutation Detection by Solid-Phase Chemical Cleavage of Mismatches Using Radioactive Probes.

Scanning the Ocular Albinism 1 (OA1) Gene for Polymorphisms in Congenital Nystagmus by DHPLC

Background: Nystagmus is common to all types of albinism. Some subjects with nystagmus lack convincing signs of albinism, have no other visual pathway disease, and are classified as possessing congenital idiopathic nystagmus (CN). It has been postulated that CN may be a form of ocular albinism. Methods: The presence of nystagmus, iris transillumination, and visual acuity were recorded in 39 CN and albino patients and their families. Physical characteristics were also noted. DNA from buccal swabs was obtained for use in denaturing high performance liquid chromatography (DHPLC) and chemical cleavage of mismatch (CCM) to scan several hotspots for X-linked ocular albinism (OA1) mutations. Results: Two previously reported polymorphisms were confirmed: neither was found to be a causative mutation. Conclusion: No correlation was identified between nystagmus and OA1.

Chemical cleavage reactions of DNA on solid support: application in mutation detection

BackgroundThe conventional solution-phase Chemical Cleavage of Mismatch (CCM) method is time-consuming, as the protocol requires purification of DNA after each reaction step. This paper describes a new version of CCM to overcome this problem by immobilizing DNA on silica solid supports.ResultsDNA test samples were loaded on to silica beads and the DNA bound to the solid supports underwent chemical modification reactions with KMnO4 (potassium permanganate) and hydroxylamine in 3M TEAC (tetraethylammonium chloride) solution. The resulting modified DNA was then simultaneously cleaved by piperidine and removed from the solid supports to afford DNA fragments without the requirement of DNA purification between reaction steps.ConclusionsThe new solid-phase version of CCM is a fast, cost-effective and sensitive method for detection of mismatches and mutations.

Mutation detection in factor VIII cDNA from lymphocytes of hemophilia A patients by solid phase fluorescent chemical cleavage of mismatch.

Defects of the factor VIII gene causes (f8) hemophilia A, an hemorrhagic X-linked disorder. The factor VIII gene is 186 kb long with 26 exons, varying from 69 bp (exon 5) to 3106 bp (exon 14) (). The factor VIII mRNA is 9028 bases in length with a coding region of 7053 nucleotides ().

Aberrant dimerization of von Willebrand factor as the result of mutations in the carboxy-terminal region: identification of 3 mutations in members of 3 different families with type 2A (phenotype IID) von Willebrand disease

AbstractThe 3′ end of the VWF gene was screened in the affected members of 3 different families with type 2A (phenotype IID) von Willebrand disease (vWD). Exons 49 to 52 of the VWF gene were amplified and screened for mutations by chemical cleavage mismatch detection. Mismatched bands were detected in exon 52 of 2 patients and in exon 51 of a third patient. Using direct DNA sequencing, a heterozygous G8562A transition leading to a Cys2008Tyr substitution was found in all the patients in family 1, and a T8561A transversion leading to a Cys2008Ser substitution was found in both patients from family 2. In a patient from a third family, an 8-base deletion from nucleotide 8437 to 8444 was identified in exon 51. The 2 mutations in exon 52 were reproduced by in vitro site-directed mutagenesis of full-length von Willebrand factor (vWF) cDNA and transiently expressed in COS-7 cells. The corresponding recombinant VWFs for these 2 mutations exhibited the typical aberrant vWF:Ag multimer pattern seen in the plasma of the patients. These 3 mutations demonstrate the importance of other carboxy-terminal cysteines in addition to the reported Cys2010 residue, in the normal dimerization of vWF, and their essential role in the assembly of normal multimeric vWF.

Detection of p53 mutations via chemical cleavage of mismatches: fundamental aspects of the methodology and further perspectives

Detection of p53 mutations via chemical cleavage of mismatches: fundamental aspects of the methodology and further perspectives

Screening of male breast cancer and of breast-ovarian cancer families for BRCA2 mutations using large bifluorescent amplicons.

41 breast cancer or breast-ovarian cancer families, including 12 families with at least one affected first-degree male relative, were screened for mutations in the BRCA2 gene. Mutations had not been found in the BRCA1 gene of these families. Chemical cleavage of Mismatch was used to identify nucleotide changes within large PCR products (average size 1.2 kb) that carried strand-specific fluorescent end-labels. 15 amplicons were sufficient to scan 18 exons, including the large exon 11. The remaining 9 small exons were examined by Denaturing Gradient Gel Electrophoresis. The high sensitivity of this approach was documented by the detection, in these 41 patients, of all 9 exonic single nucleotide polymorphisms reported with heterozygosity >0.1. Truncating BRCA2 mutations were found in 7 of the 41 families. 3 of them were in the group of 12 families comprising cases of male breast cancer. Since the methods used here have no bias for particular types of mutations, these data confirm the high proportion of frameshifts among mutations in BRCA2. However, relevant single nucleotide substitutions were also found: one resulting in a stop codon and another one, present in a male patient, was the previously reported change Asp2723His, that affects a highly conserved region of the BRCA2 protein. This study indicates a BRCA2 contribution of 10% (95% CI 2.5–17.5) to our original cohort of 59 breast-ovarian cancer families, whereas the contribution of BRCA1 had been estimated at 46% (95% CI 33–59). © 2001 Cancer Research Campaign http://www.bjcancer.com

Evaluation of DHPLC in the analysis of hemophilia A

The manifestation of hemophilia A, a common hereditary bleeding disorder in humans, is caused by abnormalities in the factor VIII (FVIII) gene. A wide range of different mutations has been identified and provides the genetic basis for the extensive variability observed in the clinical phenotype. The knowledge of a specific mutation is of great interest as this may facilitate genetic counseling and prediction of the risk of anti-FVIII antibody development, the most serious complication in hemophilia A treatment to date. Due to its considerable size (7.2 kb of the coding sequence, represented by 26 exons), mutation detection in this gene represents a challenge that is only partially met by conventional screening methods such as denaturing gradient gel electrophoresis (DGGE) or single stranded conformational polymorphism (SSCP). These techniques are time consuming, require specific expertise and are limited to detection rates of 70–85%. In contrast, the recently introduced denaturing high performance liquid chromatography (dHPLC) offers a promising new method for a fast and sensitive analysis of PCR-amplified DNA fragments. To test the applicability of dHPLC in the molecular diagnosis of hemophilia A, we first assessed a cohort of 156 patients with previously identified mutations in the FVIII gene. Applying empirically determined exon-specific melting profiles, a total of 150 mutations (96.2%) were readily detected. Five mutations (3.2%) could be identified after temperatures were optimized for the specific nucleotide change. One mutation (0.6%) failed to produce a detectable heteroduplex signal. In a second series, we analyzed 27 hemophiliacs in whom the mutation was not identified after extensive DGGE and chemical mismatch cleavage (CMC) analysis. In 19 of these patients (70.4%), dHPLC facilitated the detection of the disease-associated nucleotide alterations. From these findings we conclude that the dHPLC technology is a highly sensitive method well suited to the molecular analysis of hemophilia A.

Using CCM and DHPLC to detect mutations in the glucocorticoid receptor in atherosclerosis: a comparison

Growing evidence suggests that restenosis may be caused by a failure in growth inhibitory and apoptotic systems that would normally mediate lesion regression. One such inhibitory system is the glucocorticoid receptor. This paper develops, assesses and compares chemical cleavage of mismatch (CCM) and denaturing high-performance liquid chromatography (DHPLC) for their utility in detecting mutations in this system. The results of the two methods correlated in 74% of cases in a cohort of endarterectomy patients studied by these two methods.

Chemical mismatch cleavage analysis by capillary electrophoresis with laser-induced fluorescence detection.

Chemical mismatch cleavage analysis by capillary electrophoresis with laser-induced fluorescence detection.

Chemical cleavage of mismatches in heteroduplexes of the rpoB gene for detection of mutations associated with resistance of Mycobacterium tuberculosis to rifampin.

In order to detect mutations in the core region of the RNA polymerase B (rpoB) subunit gene of Mycobacterium tuberculosis that are known to be associated with resistance to rifampin, we applied rapid chemical cleavage of mismatches (CCM) to heteroduplexes formed between the DNA of M. tuberculosis H37Rv and strains resistant to rifampin. DNA fragments amplified from normal and mutant rpoB genes by polymerase chain reaction were mixed, denatured and re-annealed to create heteroduplexes containing mispaired bases reactive to modification by hydroxylamine (cytosine mismatches) or osmium tetroxide (thymine mismatches) and cleavage of DNA by piperidine at the position of modified base. The cleaved products and the heteroduplexes were separated by polyacrylamide-urea gel electrophoresis and detected by autoradiography. The position of mutations was confirmed by DNA sequencing of the amplified DNA fragments. The results suggest further applicability of the CCM method as a means to screen M. tuberculosis isolates for mutations associated with drug resistance.

Identification of germline missense mutations and rare allelic variants in theATM gene in early-onset breast cancer

Epidemiological studies have shown an increased risk of breast cancer in obligate ataxia telangiectasia (A-T) heterozygotes. We analyzed 100 samples from young breast cancer patients for mutations in ataxia-telangiectasia mutated (ATM), the gene responsible for the autosomal recessive condition, A-T, to determine whether A-T heterozygosity predisposes such individuals to develop breast cancer. These patients were selected from families with a moderate or absent family history of breast cancer and included a subset of 16 radiosensitive patients. Forty-four germline sequence variants were detected by fluorescent chemical cleavage of mismatch of RT-PCR products. These included seven rare variants found in nine patients (three described for the first time), but no truncating mutations. Although three variants were detected in the radiosensitive subset, this was not statistically significant compared to the nonradiosensitive group. One variant, G2765S, is likely to be a missense mutation, but the other six variants probably represent rare polymorphisms. However, five of the seven rare germline variants detected showed loss of heterozygosity of the wild-type ATM allele for one or more markers close to the ATM locus in matched tumor DNA. This high rate of somatic inactivation of ATM may indicate either that these rare variants play a role in breast cancer development or alternatively that a neighboring tumor suppressor gene is important for tumorigenesis. We found germline truncating ATM mutations to be rare in these young breast cancer patients and therefore they are unlikely to play a role in the etiology of their disease. Genes Chromosomes Cancer 26:286-294, 1999.

Creation of a novel donor splice site in intron 1 of the factor VIII gene leads to activation of a 191 bp cryptic exon in two haemophilia A patients.

We have constructed a confidential U.K. database of haemophilia A mutations and pedigrees by characterizing the gene defect of one index patient in each U.K. family. Mutations were identified by screening all coding regions of the factor VIII (FVIII) mRNA, using solid-phase fluorescent chemical cleavage of mismatch and examining additional non-coding regions of the gene. Here we report two haemophilia A patients (UK 114 FVIII:C 2% and UK 243 FVIII:C < 1%) with an abnormal FVIII mRNA due to an A to G point mutation, 1.4 kb downstream from exon 1 in the FVIII gene. This mutation creates a new donor splice site in intron 1 and leads to insertion of a 191 bp novel exon in the mRNA. Haplotype analysis suggests that the mutation may have originated in a common ancestor of the two patients, who further illustrate how mRNA analysis allows higher efficiency of haemophilia A mutation detection, because their mutation would not have been identified by direct analysis of the factor VIII gene.