A sensitive scanning technology for low frequency nuclear point mutations in human genomic DNA (original) (raw)
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Development of new molecular procedures for the detection of genetic alterations in man
Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis, 1996
The Restriction Site Mutation (RSM) procedure is a DNA-based method for detecting mutations at any unselected locus. Mutations are identified as alterations of the DNA sequence at a chosen restriction site. DNA from cells exposed to mutagenic treatment is exhaustively digested with the restriction enzyme (RE). Sequences containing the mutated target site are specifically amplified using the polymerase chain reaction (PCR), whereas DNA without mutations at this site will have been cleaved and can not therefore provide a substrate for PCR. We have developed this procedure using both bacterial and mammalian cells. With bacteria, in plasmid reconstruction experiments we were able to detect mutations at a frequency of 10 -6 at an EcoRI site in the AraA locus of Salmonella typhimurium. The detection limit with an RsaI site in the lad gene of Escherichia coli was 10 -5, and we were able to detect DNA damage and repair after treatment with N-methyl-Nnitrosourea (MNU). With mammalian cells, we have detected mutations induced by ethyl methanesulphonate (EMS) at a TaqI site in the aprt gene of Chinese hamster cells. In extensive studies with normal and repair-deficient human cells, we have detected and sequenced mutations induced by UV-C or UV-B in fibroblasts and lymphoblastoid cells from repair-deficient xeroderma pigmentosum (XP) donors. Similar results were obtained at TaqI sites in three genes, hprt, c-Ha-rasl and p53. These results demonstrate that the system is able to detect and analyse mutations induced at high frequencies. In our extensive attempts to extend the work to conditions of lower mutation frequencies, we have encountered several obstacles, the most serious being false-positive mutant DNA in totally untreated cells. This appeared to be a cell-line specific phenomenon, which we have not been able to eliminate by altering conditions. We propose therefore that, at present, RSM is a suitable method for studying high mutation frequencies at different loci and could be used for mutagen testing with repair-deficient cells. As yet, however, its sensitivity and specificity is not sufficient for population monitoring.
DNA fingerprint analysis for the detection of induced mutations in mammalian cells in culture
Cytotechnology, 1996
A mutation assay in cultured mammalian cells based on the direct analysis of minisatellite DNA was developed. Band pattern variations reflect DNA alterations ranging from single base changes to complex rearrangements. By DNA fingerprinting a large number of autosomal loci throughout the human genome can be simultaneously checked, therefore minimizing the size of the samples of cell colonies to be scored in the absence ofphenotypic selection. For the mutation assay chinese hamster cells (V79) were treated with Nitrosoguanidine and 14 independent colonies were isolated and expanded. DNA fingerprints were obtained after digestion of the DNA extracted from each clone with both HinfI and Hae III, and hybridisation with both 33.15 and 33.6 probes. Twelve colonies from untreated cells were also analysed. Several differences in the band pattern of treated colonies were observed when compared with untreated cells; digestion with Hae III and hybridisation with 33.15 probe allowed the detection of the highest frequency of induced variants. The results suggest that minisatellite sequences are hypermutable sites that can be used to monitor the mutagenic potential of chemical agents directly at the DNA level, without phenotypic selection. Moreover, with the method herein decribed, it is possible to distinguish between true mutations and epimutations, such as those caused by changes in DNA methylation.
Nucleic Acids Research, 2003
Mutations cause or in¯uence the prevalence of many diseases. In human tissues, somatic point mutations have been observed at fractions at or below 4/10 000 and 5/100 000 in mitochondrial and nuclear DNA, respectively. In human populations, fractions for the multiple alleles that code for recessive deleterious syndromes are not expected to exceed 5 Q 10 ±4. Both nuclear and mitochondrial point mutations have been measured in human cells and tissues at fractions approaching 10 ±6 using constant denaturant capillary electrophoresis (CDCE) coupled with high-®delity PCR (hi®PCR). However, this approach is only applicable to those target sequences (~100 bp) juxtaposed with a`clamp', a higher-melting-temperature sequence, in genomic DNA; such naturally clamped targets represent~9% of the human genome. To open up most of the human genome to rare point-mutational analysis, a high-ef®ciency DNA ligation procedure was recently developed so that a clamp could be attached to any target of interest. We coupled this ligation procedure with prior CDCE/hi®PCR and achieved a sensitivity of 2 Q 10 ±5 in human cells for the ®rst time using an externally attached clamp. At this sensitivity, somatic mutations, each representing an anatomically distinct cluster of cells (turnover unit) derived from a mutant stem cell, may be detected in a series of tissue samples, each containing as many as 5 Q 10 4 turnover units. Additionally, rare inherited mutations may be scanned in pooled DNA samples, each derived from as many as 10 5 persons.
Nucleic Acids Research
Using a zone of constant temperature and denaturant concentration in capillary electrophoresis, we have devised a simple, rapid, and reproducible system for separating mutant from wild type DNA sequences with high resolution. Important to the success of this method, which we call Constant Denaturant Capillary Electrophoresis (CDCE), has been the use of linear polyacrylamide at viscosity levels that permit facile replacement of the matrix after each run. For a typical 100 bp fragment, point mutation-containing heteroduplexes are separated from wild type homoduplexes in less than 30 minutes. Using laser-induced fluorescence to detect fluorescent-tagged DNA, the system has an absolute limit of detection of 3 x 104 molecules with a linear dynamic range of six orders of magnitude. The relative limit of detection at present Is 3 x10-4, i.e. 105 mutant sequences are recognized among 3 x 108 wild type sequences. The new approach should be applicable to the identification of low frequency mutations, to mutational spectrometry and to genetic screening of pooled samples for detection of rare variants.
Detection of human genomic mutations by chemical single-reaction DNA sequencing
Technical Tips Online, 1997
▼The ideal DNA sequencing method should yield complete and unambiguous information in a single electrophoretic pattern and should be simple, rapid and economical. A single-lane sequencing procedure is available, based upon the dideoxy Sanger methodology. This method cannot a priori be compressed further. Chemical DNA sequencing offers the potential of absolute compression: if one could obtain unambiguous and complete sequence information in a single pattern, then several different DNAs, each labeled with a different fluorochrome, could be analyzed in the same electrophoretic lane. This procedure could be quite relevant for mass genetic screenings.
Evaluation of MutS as a tool for direct measurement of point mutations in genomic DNA
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1997
The MutEx assay is a technique that was developed to detect and map mutations. This assay takes advantage of the Escherichia coli mismatch binding protein MutS, which binds and protects mismatched, heteroduplex DNA from subsequent exonuclease digestion. The plausibility of using the MutEx assay as part of a genotypic selection scheme was investigated. Heteroduplexes were formed between mouse H-ras gene PCR products or restriction fragments that contained wild-type Ž. sequence and sequence with a single base change at codon 61 wild-type, CAA and mutant, AAA. The heteroduplexes were incubated with MutS and then treated with the exonuclease activity of T7 DNA polymerase. MutS-protected DNA sequences Ž. were amplified by PCR. When this method was linked to single nucleotide primer extension SNuPE for mutant base identification, original mutant fractions of 1 in 50 000 and above were detected. Using comparable DNA template mixtures, the sensitivity of SNuPE alone was 1 in 5 or 1 in 50, depending on the direction of SNuPE priming and the particular base being incorporated. We conclude that the MutEx assay was able to enrich the mutant sequence approximately 1000-fold and, therefore, has considerable potential as a tool for mutation detection.
Analysis of mutational spectra by denaturing capillary electrophoresis
Nature Protocols, 2008
The point mutational spectrum over nearly any 75-to 250-bp DNA sequence isolated from cells, tissues or large populations may be discovered using denaturing capillary electrophoresis (DCE). A modification of the standard DCE method that uses cycling temperature (e.g., ±5 1C), CyDCE, permits optimal resolution of mutant sequences using computer-defined target sequences without preliminary optimization experiments. The protocol consists of three steps: computer design of target sequence including polymerase chain reaction (PCR) primers, high-fidelity DNA amplification by PCR and mutant sequence separation by CyDCE and takes about 6 h. DCE and CyDCE have been used to define quantitative point mutational spectra relating to errors of DNA polymerases, human cells in development and carcinogenesis, common gene-disease associations and microbial populations. Detection limits are about 5 3 10 À3 (mutants copies/total copies) but can be as low as 10 À6 (mutants copies/total copies) when DCE is used in combination with fraction collection for mutant enrichment. No other technological approach for unknown mutant detection and enumeration offers the sensitivity, generality and efficiency of the approach described herein.
Detection of point mutations with a modified ligase chain reaction (Gap-LCR)
Nucleic Acids Research, 1995
DNA amplification systems are powerful technologies with the potential to impact a wide range of diagnostic applications. In this study we explored the feasibility and limitations of a modified ligase chain reaction (Gap-LCR) in detection and discrimination of DNAs that differ by a single base. LCR is a DNA amplification technology based on the ligation of two pairs of synthetic oligonucleotides which hybridize at adjacent positions to complementary strands of a target DNA. Multiple rounds of denaturation, annealing and ligation with a thermostable ligase result in the exponential amplification of the target DNA. A modification of LCR, Gap-LCR was developed to reduce the background generated by target-independent, blunt-end ligation. In Gap-LCR, DNA polymerase fills in a gap between annealed probes which are subsequently joined by DNA ligase. We have designed synthetic DNA targets with single base pair differences and analyzed them in a system where three common probes plus an allelespecific probe were used. A single base mismatch either at the ultimate 3' end or penultimate 3' end of the allele specific probe was sufficient for discrimination, though better discrimination was obtained with a mismatch at the penultimate 3' position. Comparison of Gap-LCR to allele-specific PCR (ASPCR) suggested that Gap-LCR has the advantage of having the additive effect of polymerase and ligase on specificity. As a model system, Gap-LCR was tested on a mutation in the reverse transcriptase gene of HIV, specifically, one of the mutations that confers AZT resistance. Mutant DNA could be detected and discriminated in the presence of up to 10 000-fold excess of wild-type DNA.
Application of constant denaturant capillary electrophoresis (CDCE) to mutation detection in humans
Genetic Analysis: Biomolecular Engineering, 1999
Constant denaturant electrophoresis is a DNA separation technique based on the principle of cooperative melting equilibrium. DNA sequences with distinct high and low melting domains can be utilized to separate and identify molecules differing by only one base pair in the lower melting domain. Combined with capillary gel electrophoresis and when coupled with high fidelity DNA amplification, this approach can detect mutants at a fraction of 10 − 6. Modifications to the capillary elecctrophoretic system have also increased DNA loading capacity which allows for analysis of rare mutations in a large, heterogeneous population such as DNA samples derived from human tissues. Employment of this technology has determined the first mutational spectrum in human cells and tissues in a mitochondrial sequence without phenotypic selection of mutants.