Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Determination of the primary structure of abnormal Hbs on the basis of DNA sequencing of the globin gene obtained from a carrier of abnormal Hb was performed. DNA obtained from the leukocytes of the peripheral blood was amplified by the polymerase chain reaction (PCR) using the proper amplification primer set. Amplified DNA was digested with two different restriction endonucleases and cloned to vector M 13 mp 18 or mp 19, which had been digested with the same enzymes. DNA sequencing was done by the dideoxy chain termination method using T 7 DNA polymerase, and the abnormal Hbs whose primary structure was determined were as follows: Hb Fukuoka [beta 2 His(CAC/T)----Tyr(TAT)], Hb Machida [beta 6 Glu(GAG)----Gln (CAG)], Hb Hope [beta 136 Gly(GGT)----Asp(GAT)], Hb Hiroshima [beta 146 His(CAC)----Asp(GAC)] and Hb Kodaira [beta 146 His(CAC)----Gln(CAA)]. This method for determining the primary structure of abnormal Hbs might be more effective than the ordinary method, which involves amino acid analysis and amino acid sequencing of the abnormal peptide obtained from abnormal Hb.
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PMID:[Structural analysis of abnormal hemoglobin by the polymerase chain reaction (PCR) of genomic DNA]. 223 67

In order to study the conversion of UV lesions into frameshift and base substitution mutations, M13mp2 phage DNA was altered by the addition of extra pyrimidines, or by construction of a nonsense codon preceded by a run of pyrimidines within the beta-galactosidase complementing region. The normal sequence 5' GTC GTT TTA CAA 3' was changed to GTC GTT T TTA CAA (MIDT) or GTC GTT C TTA CAA (MIDC) to study frameshifts and to GTC GTT CTT TAA (OCHRE) to study reversion of the ochre (TAA) codon. Escherichia coli pol I Kf and T7 DNA polymerase mutant enzymes devoid of 3'-->5' exonuclease activity produced UV-induced revertants at higher frequency than did their exonuclease proficient counterparts. Removal of cyclobutane dimers with photolyase before in vitro synthesis did not greatly affect mutant frequency although such treatment led to significantly increased DNA synthesis by the wild-type T7 DNA polymerase on UV-irradiated substrate. Reversions of the in frame ochre sequence GTT CTT TAA produced by the delta 28 T7 DNA polymerase were mainly by base substitution in the TAA codon. About half of the E. coli Kf exo- enzyme ochre revertants had a TTA deletion. Five mutant T7 DNA polymerases with varying exonuclease activity gave revertant frequencies that correlated better with published values of enzyme velocity than with exonuclease activity or with measured bypass synthesis. Our data indicate that loss of proofreading activity increases the frequency of UV-induced frameshifts, but lack of such activity is not sufficient for their production. We suggest that frameshifts occur more frequently when nucleotide addition opposite the lesion is slow. The same lesion can give rise to a different spectrum of mutations depending on the polymerase.
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PMID:Production of UV-induced frameshift mutations in vitro by DNA polymerases deficient in 3'-->5' exonuclease activity. 802 6

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 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 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 50000 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.
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PMID:Evaluation of MutS as a tool for direct measurement of point mutations in genomic DNA. 910 Aug 51

The detection of rare mutations has many important applications, including risk assessment of drugs and chemicals, measuring environmental exposures to genotoxins, and cancer cell detection. A sensitive genotypic selection method has been developed that combines two different mutant allele selection techniques, MutEx enrichment and allele-specific competitive blocker PCR (ACB-PCR). This method was developed and evaluated for the detection of a CAA --> AAA mutation at codon 61 of the mouse H-ras gene. The MutEx enrichment is based on MutS binding to a mismatched basepair in heteroduplex DNA. The bound MutS protects the mutant allele from degradation during subsequent exonuclease treatment. ACB-PCR preferentially amplifies a mutant allele in a PCR reaction using a primer that has more mismatches to the wild-type allele than the mutant allele. By combining these two approaches, the codon 61 mutation was detected at mutant fractions as low as 1 in 10(7). This sensitivity was achieved with the thermostable Thermus aquaticus MutS protein but not the Escherichia coli MutS protein. Using the combined approach, the average Pfu DNA polymerase error rate +/- the standard error of the mean for this particular basepair was estimated to be 8 +/- 3 x 10(-7) errors per duplication. The results indicate that MutEx/ACB-PCR is among the most sensitive genotypic selection methods for the detection of mutation.
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PMID:Detection of basepair substitution mutation at a frequency of 1 x 10(-7) by combining two genotypic selection methods, MutEx enrichment and allele-specific competitive blocker PCR. 981 34

A modified allele-specific competitive blocker PCR (ACB-PCR) has been developed as an approach for genotypic selection, the detection of a rare mutant allele based solely upon its altered nucleotide sequence. ACB-PCR genotypic selection operates through the preferential PCR amplification of mutant DNA using a primer that has more mismatches to the wild-type allele than the mutant allele. In addition, a blocker-primer with a 3'-terminal dideoxynucleotide and more mismatches to the mutant allele than the wild-type allele is incorporated to reduce the background and increase sensitivity. Using ACB-PCR, the CAA-->AAA base substitution at codon 61 of the mouse H-ras gene was detected regularly at mutant fractions of 10(-5). To accurately quantify the occurrence of this particular mutation, an internal amplification standard (AS) DNA was constructed. The H-ras and AS DNAs were subject to the same genotypic selection but were amplified using different upstream primers to give PCR products that can be distinguished by size. Defined mixtures of mutant and wild-type AS DNAs were used to study the effects of various components of the ACB-PCR. The concentration of dNTPs, blocker primer and Perfect Match Polymerase Enhancer, as well as the choice of thermostable DNA polymerase and annealing temperature were examined. Conditions were identified for the concurrent detection of the CAA-->AAA mutation in the H-ras and AS DNAs. Using the identified conditions, approximately equal signals were obtained from equivalent amounts of the two DNA templates over a wide range of mutant fractions (1 in 10 to 1 in 10(5)). This ACB-PCR method can be used for any application where it is necessary to quantify relatively small mutant fractions.
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PMID:Detection of a mouse H-ras codon 61 mutation using a modified allele-specific competitive blocker PCR genotypic selection method. 986 88

Methods that detect rare base substitutions within populations of DNA molecules are valuable tools for studying the DNA-damaging effects of chemicals and for pool screening for single-nucleotide polymorphisms. Allele-specific competitive blocker-polymerase chain reaction (ACB-PCR) uses a mutant-specific PCR primer with more 3'-terminal mismatches to an abundant or wild-type sequence than to a rare or mutant sequence in order to amplify specifically an allele that differs from the wild-type by a single base pair. ACB-PCR reactions include a blocker primer to reduce the amount of background signal generated from the abundant wild-type template. The nonextendable blocker primer preferentially anneals to the wild-type DNA sequence, thereby excluding the annealing of the extendable mutant-specific primer to the wild-type sequence. Inclusion of single-strand DNA binding protein in the ACB-PCR reaction and use of the Stoffel fragment of Taq DNA polymerase both significantly increase allele discrimination. The concurrent analysis of mutant fraction standards and equivalent PCR products amplified from genomic DNA samples makes ACB-PCR a quantitative method that can detect a base pair substitution in the presence of a 105-fold excess of wild-type DNA. Methods for the ACB-PCR measurement of the mouse H-ras codon 61 CAA --> AAA mutation are presented.
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PMID:Allele-specific competitive blocker-PCR detection of rare base substitution. 1550 27

In this study we present a quick and easy method for counting trinucleotide repeats by de-oxyadenosine overhang (A-overhang)-dependent repeat expansion determination (ADRED). During standard Taq DNA polymerase-based sequencing reactions, the unterminated sequencing products of short PCR fragments are tagged with a 3'-end A-overhang that is visible as an intense peak in an electropherogram; this allows for easy and precise determination of the fragment length and thus the extent of repeat expansions. ADRED has clear advantages over existing methods, because repeat numbers of both normal and pathogenic (expanded) alleles can be analyzed without using labeled primers or labeled DNA standards. Because ADRED includes a sequencing step, disease-relevant polymorphisms (e.g., CAA interruptions in spinocerebellar ataxia type 2) can simultaneously be detected.
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PMID:A-overhang-dependent repeat expansion determination (ADRED). 1900 42

DNA structural perturbations that are induced by site specifically and stereospecifically defined benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) adducts are directly correlated with mutagenesis, leading to cellular transformation. Although previous investigations had established that replication of DNAs containing N(6) -BPDE dA adducts at the second position in the N-ras codon 61(CAA) (61(2) ) resulted exclusively in A to G transitions, NMR analyses not only established the structural basis for this transition mutation but also predicted that if the adduct were positioned at the third position in the same codon, an expanded spectra of mutations was possible. To test this prediction, replication of DNAs containing C10 S-BPDE and C10 R-BPDE lesions linked through the N(6) position of adenine in the sequence context N-ras codon 61, position 3 (C10 S-BPDE and C10 R-BPDE at 61(3) ) was carried out in Escherichia coli, and these data revealed a wide mutation spectrum. In addition to A to G transitions produced by replication of both lesions, replication of the C10 S-BPDE and C10 R-BPDE adducts also yielded A to C and A to T transversions, respectively. Analyses of single nucleotide incorporation using Sequenase 2.0 and exonuclease-deficient E. coli Klenow fragment and pol II not only revealed high fidelity synthesis but also demonstrated the same hierarchy of preference opposite a particular lesion, independent of the sequence context. Primer extension assays with the two lesions at N-ras 61(3) resulted in truncated products, with the C10 S-BPDE adducts being more blocking than C10 R-BPDE lesions, and termination of synthesis was more pronounced at position 61(3) than at 61(2) for each of the lesions.
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PMID:Sequence context modulation of polycyclic aromatic hydrocarbon-induced mutagenesis. 2391 16