Detection and localization of base changes in RNA using a chemical cleavage method
Abstract
The detection of base changes in DNA and RNA is of central importance in genetic research. Mismatched cytosines and thymines in heteroduplex DNA molecules show increased chemical reactivity with hydroxylamine and osmium tetroxide, respectively, and the DNA can then be specifically cleaved at the modified nucleotides. We show here that mismatched cytosines and thymines can be detected and located directly in RNA: DNA heteroduplex molecules. In order to detect guanosine and adenosine base changes the complementary cDNA strand must be analyzed. In addition, the sensitivity of the technique can be increased by employing the polymerase chain reaction. To test the fidelity of this method a number of known or predicted mutations were analyzed. These include single point mutations in the human collagen α1(I) and rat phenylalanine hydroxylase mRNA, two engineered point mutations in a mouse collagen α1(I) mRNA, and a deletion in a human collagen α2(I) mRNA. All known base changes were detected and correctly localized. In addition, the predicted base changes were confirmed.
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Cited by (21)
Osteogenesis imperfecta (OI) is a group of inherited disorders characterized by a predisposition to bone fracturing, and usually resulting from mutations in the genes encoding type I collagen. This report describes the molecular defects in a patient with type II OI and another with type III OI. These patients were demonstrated to possess point mutations resulting in glycine → arginine substitutions within the triple helical domain of the α1(I) or α2(I) collagen polypeptide chain. The defect in the type II OI patient affected residue 211 of the α1(I) triple helical domain, and constitutes the most amino-terminal lethal glycine → arginine substitution described to date. The substitution in the type III OI patient affected residue 427 of the α2(I) triple helical domain. Both defects were informative in that they identified the regions of the α1(I) and α2(I) collagen chains in which the phenotypes associated with glycine → arginine substitutions undergo a transition between lethal and nonlethal forms, thereby allowing a more reliable prognosis of disease severity. The histological examination of bone from these patients revealed striking abnormalities in the quantity and organization of mineralized bone structures, compared with age-matched controls. Although the patients were differently classified, no major differences in the magnitude of bone architectural changes could be perceived, consistent with the presence of their defects near a common phenotypic transition. The results are compatible with there being a gradient in severity between OI types II and III, and that parameters external to the gene mutations might account for the survival differences in the 2 cases presented in this study.
Identification of type I collagen gene polymorphisms: Tolerance of sequence variation at an α2(I) Helix Y position
1994, Matrix BiologyThis study has examined the frequency and distribution of polymorphisms in the type 1 collagen coding sequences. RNA from a group of human skin fibroblast cell lines, was analyzed by the chemical cleavage mismatch detection method using hydroxylamine, a reagent specific for C base mismatches, and overlapping cDNA probes covering the entire preproα1(I) and preproα2(I) coding regions. Mismatches were detected at only two nucleotide positions, one in each of the type I collagen sequences, suggesting that polymorphisms are relatively rare within these cDNAs. cDNA sequence analysis demonstrated that the preproα1(I) mismatch, detected in only one cell line, was due to a sequence polymorphism involving the wobble position of the codon for arginine residue 59 within the amino-propeptide globular subdomain of the proα1(I) chain and not resulting in a change in the polypeptide primary structure. In contrast, the preproα2(I) mismatch, detected in 6 of the 16 cell lines, was shown to arise from a sequence polymorphism affecting the identity of Y-position residue 459 of the α2(I) triple helical domain, resulting in an alanine/proline dimorphism at this position. This study is the first to identify a type I collagen coding sequence polymorphism resulting in an alteration at the level of the amino acid sequence. The data suggest that at least some α1(I) and α2(I) helix Y positions may be tolerant of sequence variation, particularly if the replacing amino acid is proline, a residue involved in stabilizing the collagen triple helix.
The use of chemical reagents in the detection of DNA mutations
1993, Mutation Research - Fundamental and Molecular Mechanisms of MutagenesisAs the analysis of the human genome proceeds at an ever-increasing pace, many genes have been identified which are the site for mutations responsible for inherited diseases. The identification of the mutations within these genes has become a major application of molecular biology technologies, and to this end a number of mutation detection systems have been developed for use in diagnostic and research laboratories. The uses of these mutation detection systems are in the diagnosis of inherited disease (both prenatal and neonatal) and in an understanding of the function of the affected protein by cataloguing the range of mutations. Two of these mutation detection systems are reviewed here. Both rely on chemical modification of mismatched nucleotides, by either carbodiimide or hydroxylamine and osmium tetroxide. The methods are termed the carbodiimide (CDI) and the Chemical Cleavage of Mismatch (CCM) methods. The history and evolution of the methods is tracked, illustrating the way in which they developed, both as suitable technology became available (for example, the polymerase chain reaction) and as a result of a specific need. The current methodologies are briefly discussed, followed by a discussion of their applications, especially in the realm of disease mutation detection.
Current methods of mutation detection
1993, Mutation Research - Fundamental and Molecular Mechanisms of MutagenesisMutation detection is important in all areas of biology. Detection of unknown mutations can involve sequencing of kilobases of DNA, often in many patients. This has lead to the development of methods to screen DNA for mutations as well as methods to detect previously described mutations. This review discusses current methods used for such purposes with special emphasis on genetic diseases of humans. However, savings can be made by similar means in other areas of biology where repetitive or extensive sequencing for comparative purposes needs to be done. This review covers the methods used for detection of unknown mutations, namely the ribonuclease, denaturing gradient-gel electrophoresis, carbodiimide, chemical cleavage, single-strand conformation polymorphism, heteroduplex and sequencing methods. Once mutations have been defined they can be searched for repeatedly by methods referred to as diagnostic methods. Such methods include allele-specific oligonucleotide hybridization, allele-specific amplification, ligation, primer extension and the artificial introduction of restriction sites. We can now choose from a range of excellent methods, but the choice will usually depend on the background of the laboratory and/or the application in hand. Screening methods are evolving to more satisfactory forms, and the diagnostic methods can be automated to screen whole populations inexpensively.
[19] Chemical Cleavage of Mismatch to Detect Mutations
1993, Methods in EnzymologyThe chemical cleavage of mismatch (CCM) method allows mutation sites to be detected in kilobase-length pieces of nucleic acids. A screening method such as this obviates the need to sequence the lengths of DNA to determine mutation sites. The CCM method is based on chemical and not enzymatic reactions and therefore on the problems often encountered in molecular biology. However, care must be taken with CCM in correct solution preparation. Overreaction by piperidine is sometimes seen in chemical cleavage. This is indicated by the presence of a ladder of cleavage products at each base position. In the case of diploid organisms, heteroduplex molecules are formed if self-DNA is heated and annealed, when a heterozygous mutation is present. The mutation can then be identified by CCM. If a homozygous mutation is present, CCM with self-DNA cannot identify the mutation. In this way, heterozygous and homozygous mutations can be distinguished. In conclusion, chemical cleavage offers a reliable screening method for mutation detection.
GeneScreen: A program for high-throughput mutation detection in DNA sequence electropherograms
2011, Journal of Medical Genetics