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Results for “DNA Mutational Analysis”

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PubMed1994

High throughput and economical mutation detection and RFLP analysis using a minimethod for DNA preparation from whole blood and acrylamide gel electrophoresis.

We report a simple, rapid, and high throughput method which allows the simultaneous processing of multiple whole blood samples for routine DNA purification and analysis. The method is based on a microscale DNA preparation and digestion using minimal amounts of reagents and handling. The amount of material necessary for a Southern blot analysis (5-7 micrograms) is obtained from 200 microliters of whole blood. All steps involved in DNA preparation and restriction digestion are processed in a single 1.5-ml Eppendorf tube. DNA preparation is performed using a salting out procedure with a proteinase K digestion step but no phenol/chloroform extraction. Restricted fragments are separated by electrophoresis through polyacrylamide slab gels followed by electrotransfer to nylon membranes. By varying the electrophoresis parameters (V/cm or duration), fragments of interest up to 12 kb length can be separated with high resolution. At least 80 samples can be processed at once per DNA preparation, and multiples of this number depend on the available equipment. This economical and rapid method allows routine DNA analysis for mutation or RFLP detection to be performed on a large scale which is a mandatory feature in any DNA-based population screening program. In addition, the DNA purified by the minimethod can be used as an economical source for PCR analysis.

Blotting, Southern↗

PubMed2003

The de novo Q167K mutation in the POU1F1 gene leads to combined pituitary hormone deficiency in an Italian patient.

The POU1F1 gene encodes a transcription factor that is important for the development and differentiation of the cells producing GH, prolactin, and TSH in the anterior pituitary gland. Patients with POU1F1 mutations show a combined pituitary hormone deficiency with low or absent levels of GH, prolactin, and TSH. Fourteen mutations have been reported in the POU1F1 gene up to now. These genetic lesions can be inherited either in an autosomal dominant or an autosomal recessive mode. We report on the first Italian patient, a girl, affected by combined pituitary hormone deficiency. The patient was found to be positive for congenital hypothyroidism (with low TSH levels) at neonatal screening. Substitutive therapy was started, but subsequent growth was very poor, although psychomotor development was substantially normal. Hospitalized at 10 mo she showed hypotonic crises, growth retardation, delayed bone age, and facial dysmorphism. In addition to congenital hypothyroidism, GH and prolactin deficiencies were found. Mutation DNA analysis of the patient's POU1F1 gene identified the novel Q167K amino acid change at the heterozygous level. The highly conserved Q167 residue is located in the POU-specific domain. No mutation was detected in the other allele. DNA analysis in the proband's parents did not identify this amino acid substitution, suggesting a de novo genetic lesion. From these data it can be hypothesized that the Q167K mutation has a dominant negative effect.

Amino Acid Sequence↗

PubMed1998

Clinical and laboratory findings in referrals for mitochondrial DNA analysis.

BACKGROUND: Increasingly, mutations of mitochondrial DNA (mtDNA) are being considered when investigating the aetiology of neurological diseases in childhood. However, they are often difficult to predict clinically. METHOD: Mitochondrial DNA analysis was carried out on 190 children from 1992 to 1996. Most patients were screened for large scale rearrangements and point mutations at nucleotide positions 3243, 3271, 8344, and 8993. RESULTS: Mutations were found in only 15 patients (7.9%) and were either large scale rearrangements (seven patients) or point mutations at nucleotide position 3243 (eight patients). Other point mutations were screened for depending on the clinical picture. The age of symptom onset was significantly older in children with an mtDNA mutation (mean 7.0 years) compared with children without a mutation (mean 2.8 years). Neither Leigh's syndrome (28 cases) nor severe infantile lactic acidosis (12 cases) was associated with mtDNA mutation. Only three clinical features were significantly associated with an mtDNA mutation: progressive external ophthalmoplegia, myopathy, and pigmentary retinopathy. Family history was valuable: the point mutation at nucleotide 3243 (but not the large scale rearrangements) was associated with maternal inheritance; and consanguinity was not associated with mtDNA mutations. The only investigation that provided specific evidence of an underlying mtDNA mutation was histochemical staining of muscle biopsy specimens. The large scale mutations associated with Kearns-Sayre syndrome and progressive external ophthalmoplegia were found in DNA from muscle only, not leucocyte DNA; whereas point mutations were found in leucocyte DNA. CONCLUSIONS: Even among children seen at a neurogenetic referral centre, mtDNA mutations were very uncommon. Muscle biopsy was the only investigation to provide evidence of mtDNA abnormality.

Acidosis, Lactic↗

PubMed2000

Comparative sequence analysis (CSA): a new sequence-based method for the identification and characterization of mutations in DNA.

Direct sequencing analysis is largely used to confirm and characterize mutations previously detected by more rapid tests. We have developed a method-Comparative Sequence Analysis (CSA)-that simplifies the analysis of sequencing data facilitating its use as a first screen for mutation detection. Sequence data were split into their component electrophoretograms and the use of a size standard enabled equivalent traces from different individuals to be overlaid. This allowed simple and rapid visual analysis of the results. Using this technique in a blind study, we tested 576 samples for mutations in the Von Hippel-Lindau tumor suppressor gene, VHL. We were able to identify and characterize all 78 known mutations present within the sample set (100% sensitivity and specificity).

DNA Mutational Analysis↗

PubMed1987

DNA sequence analysis of the mutational specificity of u.v. light in the SUP4-o gene of yeast.

We have characterized mutations induced in the SUP4-o gene of Saccharomyces cerevisiae by u.v. irradiation. Mutants were selected following treatment with 60 J/m2 u.v. light which reduced cell survival to 10% and increased the SUP4-o mutation frequency 100-fold above background. DNA sequence analysis of 120 mutants revealed that u.v. induced all types of base substitutions, although transitions, in particular G:C----A:T events predominated. In addition, a small number of single base pair deletions and double mutations, occurring in tandem or separated by a few base pairs, were recovered. The base pair substitutions were not distributed randomly in the SUP4-o gene and, with one exception, were all located at sites of adjacent pyrimidines, suggesting that they were targeted by u.v. photolesions. A substantial fraction of the mutations were detected at hotspots for u.v. mutagenesis. The majority of changes occurred at the 3' base of dipyrimidine sequences where both cyclobutane dimers and [6-4]-photoproducts could form. Approximately one-third of the induced base substitutions were found at potential pyrimidine dimer sites where [6-4]-photoproducts would be expected to occur rarely. The possible origins of the induced mutations and the role of cyclobutane dimers as premutational u.v. lesions in yeast are considered.

Base Sequence↗

PubMed2006

Detection and quantification of the age-related point mutation A189G in the human mitochondrial DNA.

Mutation analysis in the mitochondrial DNA (mtDNA) control region is widely used in population genetic studies as well as in forensic medicine. Among the difficulties linked to the mtDNA analysis, one can find the detection of heteroplasmy, which can be inherited or somatic. Recently, age-related point mutation A189G was described in mtDNA and shown to accumulate with age in muscles. We carried out the detection of this 189 heteroplasmic point mutation using three technologies: automated DNA sequencing, Southern blot hybridization using a digoxigenin-labeled oligonucleotide probe, and peptide nucleic acid (PNA)/real-time PCR combined method on different biological samples. Our results give additional information on the increase in mutation frequency with age in muscle tissue and revealed that the PNA/real-time PCR is a largely more sensitive method than DNA sequencing for heteroplasmy detection. These investigations could be of interest in the detection and interpretation of mtDNA heteroplasmy in anthropological and forensic studies.

Adolescent↗

PubMed2003

Ancient DNA analysis of the delta F508 mutation.

When working with highly degraded DNA, validating the results of a slightly polymorphic system always complicates the analysis because of the difficulties in recognizing contamination and artifacts. Recognition can be greatly simplified by employing a multiplex reaction that coamplifies the fragments together with several highly polymorphic markers, for instance, short tandem repeats. In this work, we successfully included newly designed oligonucleotide primers for the detection of delta F508, the most frequent mutation causing cystic fibrosis, in the commercial AmpFlSTR Profiler Plus PCR Amplification Kit (PE Applied Biosystems). This coamplification enabled us to test the hypothesis of a heterozygote advantage associated with cystic fibrosis-specifically, higher resistance to toxin-mediated diarrheas--in a Sicilian skeletal sample of individuals who died in a cholera epidemic in 1837. The proposed method should also be suitable for the genetic characterization of other slightly polymorphic loci tested on human and animal ancient DNA; it should permit simple authentication of results by comparing the fingerprints obtained from independent amplifications repeated several times.

Cystic Fibrosis↗

PubMed1999

DNA sequence analysis of spontaneous tonB deletion mutations in a polA1 strain of Escherichia coli K12.

By DNA sequence analysis, we have determined a spectrum of 61 spontaneous mutations occurring in the endogenous tonB gene in the polA1 strain of Escherichia coli. The overall mutation frequency was approximately 2.4-fold higher in the polA1 strain and this was attributable to enhanced rates of deletion and frameshift mutations. Among 39 deletions, a hot spot (17 mutations) was detected: a 13-bp deletion presumably directed by a 3-bp repeated sequence at its end points. The remaining 22 were distributed among 19 different mutations either flanked (16/19) or not flanked (3/19) by repeated sequences. Single-base frameshifts accounted for 8 mutations of either repeated (3/8) or nonrepeated (5/8) bases among which 6 were minus one frameshift. In contrast to previous reports, we did not frequently observe a 5'-GTGG-3' sequence in the vicinity of the deletions and frameshifts. The results presented here indicated an anti-deletion and anti-frameshift role for DNA polymerase I.

Bacterial Proteins↗

PubMed1994

A common hot spot for somatic and germline mutations in rat mitochondrial DNA: analysis by fluorescence-based SSCP.

To estimate the degree of sequence heterogeneity of mtDNA, we have developed an efficient system for mutant detection using fluorescence-based single strand conformational polymorphism (F-SSCP). The F-SSCP analysis 200 clones from Wistar rat and sequencing of 2 clones from each 40 inbred Wistar rats at the age of 7 weeks detected no sequence differences, suggesting that the mtDNA sequence heterogeneity is low in young rat hearts. One of the 387 clones isolated from a Donryu rat showed a different mobility in the F-SSCP from other clones. Sequencing of the mutant clone revealed double contiguous TT-->CC transitions at nucleotide positions 15443 and 15444, compared with the normal Donryu clone. The nucleotide at 15444 was different between the Wistar and Donryu rats, indicating that this is the site of germline mutation. These results suggest that the somatic and germline mutations can occur at a common hot spot.

Animals↗

PubMed1999

Evaluation of the protein truncation test and mutation detection in the NF1 gene: mutational analysis of 15 known and 40 unknown mutations.

The neurofibromatosis type 1 (NF1) gene located at 17q 11.2 contains 60 exons and spans 350 kb of genomic DNA. Mutation analysis has been hampered by the large size of the gene, the high rate of new mutations, a lack of mutational clustering and the presence of numerous homologous loci. Mutation detection methods based on the direct analysis of a gene's RNA transcript permit the rapid screening of large multi-exonic genes. However, the detection of frame-shift or nonsense mutations can be limited by instability of the mutant mRNA species due to nonsense-mediated decay. In order to determine the frequency of this allelic exclusion, total lymphocyte RNA was analysed from 15 NF1 patients with known truncating mutations and a panel of 40 NF1 patients with unknown mutations. The level of expression of the mutant message was greatly reduced in 2 of the 15 samples (13%), and 3 of the 18 informative samples from the panel of 40. A coupled reverse-transcription polymerase chain reaction and protein truncation test method was subsequently applied to screen RNA from the panel of 40 unrelated NF1 patients. Aberrant polypeptide bands were identified and characterised in 21 samples (53%). The mutations identified were 479del107;ins31, 495delTGTT, 1127delTGAT, R416X, R440X, 1446del 62, 1541delAG, 2252del 74, 2537insTG, 3456delACTC, R1276X, R1362X, 5749ins171, 6084del280, 6487insA, R2214X, 6791insA, 6858del141, 7458delC, 7676 2A-G and 8081delC. These mutations were uniformly distributed across the gene and 14 represent novel changes that contribute to the germline mutational spectrum of the NF1 gene.

Alleles↗

PubMed2006

Cockayne syndrome type A: novel mutations in eight typical patients.

Cockayne syndrome is a rare autosomal recessive neurodegenerative disorder. It is considered to be a heterogeneous condition based on complementation in cell fusion studies, with two major forms, namely CS-A and CS-B. CKN1 is the gene responsible for CS-A, whose mutations disrupt the transcription-coupled repair system of the actively transcribed DNA. Mutation analysis of the CKN1 gene in eight typical CS-A Brazilian patients from six families showed a gene alteration in all of them. We found a total of five novel mutations that were absent from healthy control subjects. Six affected subjects were simple homozygotes and two affected siblings were each compound heterozygotes. While the findings extend the range of mutations in CS-A, there is no obvious genotype-phenotype correlation across the mutational spectrum.

Brazil↗

PubMed2005

[DNA-analysis in hereditary cancer: the importance of a reliable family history].

Three patients, a 45-year-old man, a 51-year-old woman and a 43-year-old woman, wanted to know whether they had a hereditary predisposition for cancer. The family of patient A fulfilled the clinical diagnostic criteria for hereditary non-polyposis colorectal carcinoma (HNPCC). The family of patient B fulfilled the clinical diagnostic criteria for hereditary breast/ovarian cancer (HBOC). The family of patient C did not completely fulfil the criteria for HBOC since only two family members had a confirmed diagnosis of breast cancer. In all three families, DNA-mutation analysis was performed. In families A and B no mutation was found. However, based on the family history, the diagnosis of hereditary cancer was made and recommendations for surveillance were given. After extensive counselling, one member of family B eventually decided to have prophylactic surgery performed. A few years later, a pathogenic mutation in BRCA2 was found in family B. In family C, an unclassified variant was found in BRCA1. Further investigations in the family were not possible, due to a lack of co-operation from family members. It is important to obtain a thorough and complete family history. When DNA-analysis remains inconclusive or if an unclassified variant is found, recommendations for surveillance will be based on this family history.

Adult↗

PubMed1999

Reduced expression of CDKN2a/P16INK4a in mycosis fungoides.

Alterations in the CDKN2a gene have been demonstrated in a wide range of human tumors including hematopoietic malignancies. To verify whether altered CDKN2a expression is involved in the pathogenesis of mycosis fungoides (MF), we examined mRNA expression in 20 patients with MF by RT-PCR and dot blot hybridization. CDKN2a mRNA expression was undetectable in 5 of the 20 patients (25%), intermediate in 13 (65%) and high in 2 (10%). Immunohistochemical studies, which were performed in ten patients, revealed that in the four patients showing no mRNA, p16INK4a was expressed in <1% of neoplastic lymphocytes whereas in the four patients with an intermediate mRNA level, specific nuclear staining was present in 1-25% of tumor cells. In the two patients with high levels of CDKN2a mRNA, >25% of neoplastic lymphocytes stained positively. No direct correlation between clinicopathological and molecular findings was evident in our patients. DNA mutational analysis revealed no alterations in a total of six patients examined. Our results indicate that the lack of CDKN2a expression, as found in 25% of the patients, may have a pathogenetic role in MF even though the absence of CDKN2a mRNA was not associated with point mutations or minor gene deletions.

Adolescent↗

PubMed1999

DNA fingerprint analysis reveals differences in mutational patterns in experimentally induced rat peritoneal tumors, depending on the type of environmental mutagen.

We performed tumor DNA fingerprint analysis using the synthetic minisatellite probe S3315x2 based on the 33.15-repeat unit. The aim of the study was to investigate fingerprinting patterns of peritoneal tumors induced experimentally in Wistar rats by two carcinogens with unknown mechanism of action (crocidolite asbestos and nickel powder) and, as a positive control, benzo[a]pyrene. The carcinogens were administered intraperitoneally into rats. The banding patterns obtained with DNA from 71 peritoneal tumors were compared to the corresponding normal tissues. DNA derived from peritoneal tumors induced by the three carcinogens differed with respect to mutation frequencies and mutation patterns. The mutation frequencies in these tumors, revealed by DNA fingerprinting, were 18.2% for benzo[a]pyrene, 14.8% for crocidolite asbestos, and 40.9% for nickel powder. The alterations detected in the banding pattern of benzo[a]pyrene-induced peritoneal tumors were exclusively additional bands. On the contrary, in the DNA from asbestos-induced peritoneal tumors, only deletions of bands were observed on the autoradiographs. In the DNA from nickel-induced peritoneal tumors, both types of mutations occurred. The different mutation frequencies and mutation patterns appear to discriminate between benzo[a]pyrene, crocidolite asbestos, and nickel powder, and may be related to the mechanisms of action of these compounds.

Animals↗

PubMed1996

Detection of K-ras mutations in stools of patients with colorectal cancer by mutant-enriched PCR.

Mutant-enriched PCR was applied to the detection of mutations at codons 12 and 13 of K-ras genes in the stools of patients with colorectal cancer. Mutations were analyzed in stool samples obtained prior to surgery. Resected tumor specimens were screened for K-ras mutations by PCR-mediated RFLP analysis. Using normal stool samples, assay conditions were adjusted to optimal sensitivity and specificity. The following specimens were included in the study: 16 stool samples corresponding to carcinomas in which K-ras mutations had been identified; 7 randomly selected stool samples corresponding to carcinomas which were negative for K-ras mutations; 1 stool sample from a patient with non-Hodgkin's lymphoma. In 13 of the 16 stool samples (81%) corresponding to tumors in which K-ras mutations had been identified previously, K-ras mutations were detected. In 2 of the 7 stool samples corresponding to tumors in which K-ras mutations had not been detected by previous PCR-mediated RFLP analysis, K-ras mutations were also present. Reanalyses of the tumors corresponding to these 2 positive stool samples by mutant-enriched PCR revealed a K-ras mutation in one of the tumors. The stool and tumor of the patient with non-Hodgkins lymphoma were negative for K-ras mutations. DNA sequence analysis revealed that, for each of the K-ras mutations identified in stool samples, identical base substitutions were present in the corresponding tumor tissue. The results indicate that tumor cells harboring K-ras mutations can be detected in the stools of patients with colorectal cancer by mutant-enriched PCR with high sensitivity and specificity. Because of the simplicity of the technique, it may be suitable for screening of stool samples for mutations of the K-ras gene.

Adult↗

PubMed2006

Amplicon DNA melting analysis for mutation scanning and genotyping: cross-platform comparison of instruments and dyes.

BACKGROUND: DNA melting analysis for genotyping and mutation scanning of PCR products by use of high-resolution instruments with special "saturation" dyes has recently been reported. The comparative performance of other instruments and dyes has not been evaluated. METHODS: A 110-bp fragment of the beta-globin gene including the sickle cell anemia locus (A17T) was amplified by PCR in the presence of either the saturating DNA dye, LCGreen Plus, or SYBR Green I. Amplicons of 3 different genotypes (wild-type, heterozygous, and homozygous mutants) were melted on 9 different instruments (ABI 7000 and 7900HT, Bio-Rad iCycler, Cepheid SmartCycler, Corbett Rotor-Gene 3000, Idaho Technology HR-1 and LightScanner, and the Roche LightCycler 1.2 and LightCycler 2.0) at a rate of 0.1 degrees C/s or as recommended by the manufacturer. The ability of each instrument/dye combination to genotype by melting temperature (Tm) and to scan for heterozygotes by curve shape was evaluated. RESULTS: Resolution varied greatly among instruments with a 15-fold difference in Tm SD (0.018 to 0.274 degrees C) and a 19-fold (LCGreen Plus) or 33-fold (SYBR Green I) difference in the signal-to-noise ratio. These factors limit the ability of most instruments to accurately genotype single-nucleotide polymorphisms by amplicon melting. Plate instruments (96-well) showed the greatest variance with spatial differences across the plates. Either SYBR Green I or LCGreen Plus could be used for genotyping by T(m), but only LCGreen Plus was useful for heterozygote scanning. However, LCGreen Plus could not be used on instruments with an argon laser because of spectral mismatch. All instruments compatible with LCGreen Plus were able to detect heterozygotes by altered melting curve shape. However, instruments specifically designed for high-resolution melting displayed the least variation, suggesting better scanning sensitivity and specificity. CONCLUSION: Different instruments and dyes vary widely in their ability to genotype homozygous variants and scan for heterozygotes by whole-amplicon melting analysis.

Benzothiazoles↗

PubMed1998

Detection of APC mutations in stool DNA of patients with colorectal cancer by HD-PCR.

In most cases the analysis of DNA mutations in presence of a high excess of wild type DNA fail because of the low sensitivity of the performed method for mutation detection. Here we describe the new high-sensitive and non-radioactive HD-PCR method (for HeteroDuplex-PCR). In opposite to the conventional analytical application the heteroduplex technique is performed to preparatively separate mutated from non-mutated PCR amplified DNA fragments. We used the new method to detect mutations in the tumor suppressor gene APC in stool DNA from patients with sporadic colorectal carcinomas. Since the alteration of the APC gene occurs early in most colorectal tumors, the detection of APC mutations in fecal tumor DNA by HD-PCR may be a powerful tool in non-invasive cancer diagnostics.

Adenoma↗

PubMed1993

Molecular analysis of DNA adducts and hprt mutations produced by 6-nitrosochrysene in Chinese hamster ovary cells.

We have characterized the mutational spectrum of 6-nitrosochrysene in the hprt gene of Chinese hamster ovary (CHO-K1) cells and also examined the adducts formed by this compound in CHO-K1 cells by quantitative 32P-postlabeling analysis. Seventy percent of the identified mutations were simple basepair substitutions, and they occurred more often at A:T (14/17) than at G:C. Furthermore, 13 of the basepair substitutions at A:T had the mutated dA, the probable adducted residue, on the non-transcribed DNA strand. The preference for mutation at A:T contrasted sharply with the distribution of adducts formed by 6-nitrosochrysene: 80% of the identified adducts were with dG, while only 20% were probably formed through binding with dA. Analyses conducted with excision-repair-defective CHO-UV5 cells revealed both a preference for basepair substitution at A:T and an adduct profile that were similar to those found for repair-proficient CHO-K1 cells. However, basepair substitutions from CHO-UV5 cell mutants had the mutated dAs distributed randomly between the non-transcribed and transcribed DNA strands. The mutational spectra found for solvent control CHO-K1 and CHO-UV5 cells differed from those of the 6-nitrosochrysene-treated cultures. These findings suggest that 6-nitrosochrysene-induced mutations are targeted to DNA damage, but that 6-nitrosochrysene-derived dA adducts are much more effective at producing mutations than 6-nitrosochrysene-derived dG adducts. The extreme strand bias for mutated dAs in the CHO-K1 mutational spectrum appears to result from preferential removal of 6-nitrosochrysene-induced DNA lesions from the transcribed DNA strand.

Animals↗