Detection Of DNA Alterations Research Articles

Direct detection of deletion mutations in the yeast DEL assay using quantitative PCR (TaqMan ®)

Established mutagenesis assays measure mutant frequencies at selectable loci. These assays work by encouraging the growth of mutants to form visible colonies while suppressing the growth of non-mutants. An alternative strategy is to detect DNA alterations directly. We present an example of this latter strategy using TaqMan ® to quantify deletion mutants in mixed cultures of Saccharomyces cerevisiae strain RS112. The RS112 strain contains two heteroallelic his3 sequences that share ∼400 bp homology and are separated by ∼7 kb of plasmid DNA. Spontaneous and chemical-induced strand breaks that occur in this region are repaired by intrachromosomal recombination, resulting in the loss of the plasmid DNA and creation of a His prototroph. Ordinarily, these prototrophs are detected by growth on His− medium over 2–3 days. In this case, we used TaqMan ® to selectively detect the DNA of deletion mutants in the presence of a large excess of DNA from non-mutants. This was accomplished using primers whose annealing sites were outside the region of DNA lost due to recombination. Thus, the primers were too far apart to produce PCR products using DNA from non-mutants, but produced a robust TaqMan ® signal using DNA from deletion mutants. Spontaneous and chemical-induced recombination frequencies (RF) were measured in a series of time-course and dose–response experiments with direct-acting mutagens. Interestingly, chemical-induced increases in RF were observed within a few hours of initiation of exposure, demonstrating that deletion mutations in RS112 can be fixed soon after DNA damage occurs. The ability to measure RF at any time during treatment will be useful for additional mechanistic studies. Chemical-induced increases in RF were also observed in the absence of selective growth conditions. As such, detection of deletion mutations with TaqMan may be applicable to measurements of RFs at non-selectable loci in yeast and other species. Finally, chemical-induced RFs after 17 h exposure were similar to those observed after 3 days growth on selective medium. The TaqMan ® assay may therefore be used to screen compounds more quickly for their ability to cause deletion mutations than is currently done by plating on selective medium.

English

RAPD technique was used in this study to detect DNA band variations between both normal and abnormal male of broiler chicken based on RAPD marker. DNA polymorphisms between normal and mutant birds were detected using fifteen oligonucleiotide primers. Using these primers, DNA band loss ranged from 25 to 75%. Data demonstrated that RAPD marker could detect DNA alterations.

Stage independent molecular detection of DNA-alterations in serum- and urine of bladder cancer patients

Stage independent molecular detection of DNA-alterations in serum- and urine of bladder cancer patients

Detection of DNA Abnormalities by Arbitrarily Primed PCR Fingerprinting: Amplification of theMDM2Gene in a Mediastinum Fibrosarcoma

Arbitrarily primed PCR (AP-PCR) fingerprinting method is easy and useful for analysis of genetic alterations in anonymous chromosomal regions. We applied this technology to analysis of DNA from human primary cancers and found amplification of a DNA fragment in a mediastinum fibrosarcoma. PCR-based analysis of radiation hybrid panels following cloning and nucleotide sequence determination of the fragment revealed that it was derived from a region of chromosome 12q13-q15. In this region, theMDM2andIFNGgenes were noted as known genes that could be involved in human carcinogenesis. Southern blot analysis of genomic DNA of the tumor revealed the amplification of theMDM2gene together with the fragment locus, but not theIFNGgene. Our results demonstrated that detection of DNA alterations by AP-PCR fingerprinting without any previous knowledge of the genes and subsequent analysis of radiation hybrid panels could lead to easy identification of candidates for genes involved in carcinogenesis.

Characterization of RTG-2 Fish Cell Line by Random Amplified Polymorphic DNA

The increasing presence of genotoxic chemicals in the aquatic environment has led to the development of bothin vivoandin vitroassays for target species. The fish population represents an important level of aquatic ecosystems that can be threatened by increased environmental pollution. The authors have studied the DNA pattern of the RTG-2 fish cell line, a fibroblast-like cell line, derived from rainbow trout (Oncorhynchus mikyss), to use this cell line as anin vitrosystem to study genotoxicity by means of random amplified polymorphic DNA primers (RAPDs). A constant pattern in the DNA band is essential when an organism or cell line is used to detect DNA alterations produced by genotoxic environmental chemicals. DNA fingerprints with RAPDs were obtained for RTG-2 by testing 26 single and 70 pairwise combinations of primers. Different methods of DNA extraction (chelating resin, salting out, and phenolization), the influence of spectrometric measures at 320 nm in the 260/280 quotient to quantify DNA extracts, genomic DNA and primer concentrations, annealing temperatures, and cell line passage were studied in the cell line characterization. RAPD products were identified by agarose gel electrophoresis. The good results obtained should allow the use of this system as a possible tool for detection of the genotoxicity of aquatic pollutants.

DNA alterations detected in the progeny of paternally irradiated Japanese medaka fish (Oryzias latipes).

A nonmammalian test system for germ-cell mutagenesis has been developed by using the Japanese medaka fish. We describe a system for detecting DNA alterations in F1 progeny descended from the gamma-irradiated male medaka that uses an arbitrarily primed polymerase chain reaction and fingerprinting. A combination of these two methods has some advantages for screening changes in genomic DNA of individual progeny because this detection system can (i) screen for mutational events before embryos with dominant lethal mutations are eliminated during development and (ii) detect DNA changes in the progeny of irradiated males without functional selection and bias, such as resistance to chemicals. DNA alterations are detected as changes in patterns (i.e., band loss and/or band gain) of DNA fingerprints of progeny descended from males whose spermatozoa or spermatids were gamma-irradiated (4.75 or 9.50 Gy). We determined the frequency of gamma-irradiation-induced band loss in arbitrarily primed polymerase chain reaction fingerprints of DNA from severely malformed embryos with dominant lethal mutations and hatched viable embryos. The frequency of band loss in both dominant lethal embryos and hatched viable embryos increased with increasing gamma-ray dose, although more so in the former. We detected a new band in the fingerprints as a heritable DNA alteration but not a viability- or phenotype-affecting DNA alteration in two viable mutants recovered after gamma-irradiation experiments. A cloned amplified fragment of the new band contained a repeated sequence of p(ATGT)n.

Demethylation of a repetitive DNA sequence in human cancers.

To detect DNA alterations in unknown regions in human cancers, we have performed restriction landmark genomic scanning (RLGS) analysis of DNA isolated from cancer and normal cells. One spot with a highly intensified signal was detected in DNA from all six malignant melanoma cell lines, two of five colon cancer cell lines and one of six pancreatic cancer cell lines analyzed. In DNA from normal cells, two placentas and seven cultured lymphocytes, the signal of this spot was not intense. The DNA fragment corresponding to the spot was cloned. By nucleotide sequence analysis, the DNA fragment was revealed to be a part of a repeating unit of a 13 kbp nucleotide sequence of which 200 copies were located in chromosome 8q21. Southern blotting analysis using the cloned fragment as a probe demonstrated that the intensified signal for the DNA fragment observed in cancer cells was due to demethylation in the recognition sequence of the NotI restriction enzyme. The results suggest that marked demethylation in the repeating units might be associated with the genesis or progression of some types of cancers.

Detection of DNA alterations in human bladder tumors by DNA fingerprint analyses

DNA fingerprint analyses were used to examine the constitutional and tumor DNA from 22 bladder tumor patients. DNA alterations, such as loss of bands, new bands, and intensity shifts were observed in 10 of the 22 patients. The most frequent DNA alteration, occurring in 80% of the patients, was a complete loss of one or several bands. Fingerprint abnormalities were present both in low-malignant superficial tumors and in high-malignant invasive tumors, but were also lacking in the latter group. Apparently no relationship exists between fingerprint abnormalities and gross chromosomal aberrations or the proportion of S-phase cells as measured by flow cytometry or development of recurrent tumors during a limited observation period. Thus, whether fingerprint aberrations express genetic alterations directly involved in the malignancy potential of bladder carcinoma remains an open question.

Diagnosis of human heritable defects by recombinant DNA methods.

Recombinant DNA methods provide highly sensitive means for the detection of DNA alterations that lead to human disease mutations. In this paper I shall illustrate the approaches currently available and discuss new technologies that show promise of replacing the present methods. Medical diagnosis by means of recombinant DNA methods has an expanding role in clinical medicine.