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CZE detection of nested ARMS products. CZE of the set of five mutant (MUT, upper tracing) and six wild-type (WT, lower profile) ARMS-amplified DNA fragments for detection of 21-hydroxylase deficiency . The corresponding mutations are listed in Table 1. CZE conditions are as described in Materials and Methods. RFU, relative fluoresence units.
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A quick diagnosis of the classic form of 21-hydroxylase deficiency (simple virilizing and salt wasting) is of great importance, especially for prenatal diagnosis and treatment in pregnancies at risk. A method for simultaneous detection of common point mutations in the P450c21 B gene is here proposed by combining a nested PCR amplification refractor...
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Context 1
... nested ARMS was 90 -95 °C, allowing us to use a two-temperature amplification pro- file. With those primers we observed a good reproduc- ibility by testing this system in CAH patients and the previously genotyped parents. In all the samples ana- lyzed by this protocol, we have excellent agreement with the results obtained by ASO or sequencing. In Fig. 1 are shown all the possible peaks that we can obtain in the wild-type and mutant sets, after resolution in a 27-cm coated capillary in the presence of 30 g/L HEC as sieving liquid polymer in TBE buffer. We can see that also the polymorphism A/C in the same 655 position of intron 2 mutation is well resolved. Easy identification of the ...
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Citations
... Gel-based capillary electrophoresis and mass spectrometry, which can separate a mixture of allele-specific products generated with technologies such as oligonucleotide ligation or primer extension, have also been used as multiplex detection systems (29,30 ). ...
After the completion of the Human Genome Project, the goal for biomedical research is to apply genomic approaches for the improvement of human health (1). The study of human DNA variation promises to have a great impact on understanding how genetic factors contribute to human disease, conferring susceptibility or resistance; it is also expected to help uncover the reasons that individuals respond differentially to therapeutics.
Technologies for mutation detection can be classified into methods for the detection of unknown mutations and methods for the detection of known mutations (2). Methods for the detection of unknown mutations (3)(4)(5)(6)(7)(8), also known as screening or scanning methods, are used during the discovery phase. Once a new mutation associated with a genetic disease has been characterized and validated, it is usually detected over and over in many DNA samples by specific, cost-efficient, and easy-to-use methods (9)(10)(11)(12)(13), also known as “diagnostic methods” (14).
Typically, genotyping assays for known mutations start with sample preparation followed by amplification of the target DNA sequence containing the mutation to be tested. This latter step is most commonly accomplished by use of the PCR, but other amplification strategies are available (15)(16)(17). Subsequently, the presence or absence of the mutation is determined by allele-specific biochemical reactions. The selected allele-specific method largely determines the specificity, accuracy, and reproducibility of the genotyping assay. The discriminating power of DNA ligases (13)(15) and DNA polymerases (10)(11)(12), as well as the difference in thermodynamics between matched and mismatched DNA duplexes (9), has been extensively exploited for mutation detection. The final step of a genotyping approach is the detection of the signal resulting from the allele-specific assay and …
... As a result, a variety of strategies have been adopted to maximize specificity. These include the inclusion of a subterminal mismatches (7), the use of two allele-specific primers (8), and using nested amplification reactions with the innermost being allele specific (9). It is reasonable to state that any useful embodiment of ASPCR requires careful optimization and standardization of reaction conditions to obtain reproducibly adequate specificity. ...
A simulation of competitively primed allele-specific DNA amplification has been constructed and its behavior examined. This has shown that when the ratio of the amount of homoduplex misprime product to the total amount of amplimer is low, it increases by approximately one-fourth of the mispriming frequency with each doubling of the total amount of amplimer. When the ratio is high and reverse mispriming becomes significant, it asymptotes toward a value <0.5. An analogous simulation was carried out on conventional allele-specific DNA amplification. As expected, the ratio of the amount of amplimer in the positive and negative reactions closely approximates the mispriming frequency provided that amplification is exponential in both cases. This suggests that conventional allele-specific amplification has somewhat higher inherent specificity than competitively primed amplification. However, conventional allele-specific reactions are subject to a "catch-up" phase in which the positive reaction slows or stops, thus reducing the specificity. It was hypothesized that competitively primed reactions may be easier to optimize than conventional allele-specific reactions. This conjecture was supported experimentally. In addition, it was shown that the specificity of competitively primed reactions is a function of the degree of amplification.
Congenital adrenal hyperplasia (CAH) refers to a group of autosomal recessive genetic disorders that arise from defective steroidogenesis. The 21-hydroxylase deficiency (21OHD) is the most common form of CAH, accounting for more than 90% of cases. It is the most common disorder of sexual development (DSD) in females. The gene is encoded by CYP21A2, which is located on the short arm of chromosome 6 (6p21.3). The activity of the enzyme 21-hydroxylase, encoded by the CYP21A2 gene, is deficient, leading to an accumulation of 17-hydroxyprogesterone (17-OHP) and subsequent elevation of androgens. The three forms of 21OHD are the salt-wasting form, simple-virilizing form, and non-classical form. The first two forms are classical forms of the disease where the hallmark finding is ambiguity of the genitalia in affected female newborns. Patients with the non-classical form have normal genitalia, yet may present with signs of early sexual development and other symptoms of hyperandrogenemia such as short stature, hirsutism, acne, and impaired fertility. Hormonal testing is important in making the diagnosis of 21-hydroxylase deficiency, yet genetic testing is crucial to secure the diagnosis. More than 100 mutations have been identified caused by gene conversions, large scale gene deletions, and de novo mutations, and novel mutations are continuously being identified. Genotype-phenotype non-concordance is observed in a significant number of patients.
Congenital adrenal hyperplasia due to deficiency of steroid 21-hydroxylase (CYP21) is most frequently due to mutations that arise from the nearby CYP21 pseudogene. The mechanism involves either unequal crossing over, which deletes part of the CYP21 functional gene, or gene conversion which puts a mutation from the pseudogene into the functional gene. We have devised an assay to rapidly screen for five known mutations that are due to gene conversion within an 1,800 bp region of the CYP21 gene–I172N, V281L, Q318X, R356W, and a cluster of mutations in exon 6 (I236N, V237E, M239K). This method is based on a set of nested allele-specific polymerase chain reactions done simultaneously in one tube, for which we suggest the acronym NASA, for nested allele-specific amplification. The assay is capable of detecting the mutations individually as well as all combinations of mutations tested.
Capillary electrophoresis (CE) is electrophoresis in capillary format. It is the most efficient and versatile separation technique nowadays for analysis of small and large molecules. Clinical chemistry is a science that applies the knowledge of chemistry to the understanding of the human in health and disease. Implementation of CE technique in clinical laboratories will enhance the capability of the current clinical diagnostic system, and improve the quality of clinical testing. Owing to the nature of clinical diagnostics, the adoption of CE methods in clinical laboratories has been slow. However, CE has begun to replace some older and antiquated methods in some pioneering clinical laboratories.
This review includes a brief introduction to the CE technique (i.e. the separation principle, modes of operation, instrumentation, and practical considerations), and a summary of CE's applications in clinical chemistry (i.e. protein analysis, drug monitoring, DNA and RNA analyses, diagnosis of metabolic disorders, and single‐cell analysis), as well as the future prospects for the CE technique.
More than 90% of cases of congenital adrenal hyperplasia (CAH, the inherited inability to synthesize cortisol) are caused by 21-hydroxylase deficiency. Females with severe, classic 21-hydroxylase deficiency are exposed to excess androgens prenatally and are born with virilized external genitalia. Most patients cannot synthesize sufficient aldosterone to maintain sodium balance and may develop potentially fatal "salt wasting" crises if not treated. The disease is caused by mutations in the CYP21 gene encoding the steroid 21-hydroxylase enzyme. More than 90% of these mutations result from intergenic recombinations between CYP21 and the closely linked CYP21P pseudogene. Approximately 20% are gene deletions due to unequal crossing over during meiosis, whereas the remainder are gene conversions--transfers to CYP21 of deleterious mutations normally present in CYP21P. The degree to which each mutation compromises enzymatic activity is strongly correlated with the clinical severity of the disease in patients carrying it. Prenatal diagnosis by direct mutation detection permits prenatal treatment of affected females to minimize genital virilization. Neonatal screening by hormonal methods identifies affected children before salt wasting crises develop, reducing mortality from this condition. Glucocorticoid and mineralocorticoid replacement are the mainstays of treatment, but more rational dosing and additional therapies are being developed.
Molecular diagnosis of complex inherited disorders, population screening of genetic diseases, studies of the genetic basis of variable drug response (pharmacogenetics) as well as discovery and investigation of new drug targets (pharmacogenomics) involve screening for mutations in multiple DNA samples. Furthermore, the development of a third generation of the human genome map, based on single nucleotide polymorphisms (SNPs), requires screening for allelic variants through all of the three billion basepairs in the human genome. Thus, the need for high throughput mutation screening methods is great and is rapidly increasing. Traditional methods for mutation screening often involve slab-gel electrophoresis analyses which are laborious and difficult to automate. However, recent developments in capillary electrophoresis systems for DNA fragment analysis have made fully automated mutation screening possible and have dramatically increased the possible sample throughput. This review describes the recent advances in capillary electrophoresis of DNA and summarize the various methods for mutation screening based on this technique.
