Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

cDNA, synthesized from rabbit globin mRNA, was used in a self-priming reaction, with avian myeloblastosis virus DNA polymerase, for the synthesis of double-stranded DNA. Globin DNA ranging from about 400 to 650 base pairs was elongated with dG tails using deoxypolynucleotide transferase and was annealed to linear Escherichia coli plasmic pCR1, elongated with dC tails. Preparation of the plasmid DNA involved an enzymatic reconstruction of one EcoRI-specific site on each side of the molecule. After transformation of E. coli cells to kanamycin resistance with the hybrid molecules, bacterial clones harboring recombinant plasmids were studied for the presence of globin-specific DNA. Plasmids containing either alpha or beta rabbit globin gene sequences were obtained. There was a 4-fold excess of recombinant plasmids containing beta-globin sequences over those with alpha-globin DNA. The longest beta-globin sequences found in plasmids were about 550 to 600 pairs long, and correspond therefore to the entire beta-globin structural gene and to some of the untranslated regions. The alpha-globin sequences were 400 to 450 base pairs long. Treatment of clone pCR1betarG 19 with EcoRI endonuclease released two DNA fragments (410 and 210 base pairs) resulting from cleavage at two reconstructed external EcoRI sites and at one internal EcoRI site within the rabbit globin gene. The same treatment of pCR1alpharG 11 released one fragment. In most other recombinant plasmids studied however, no fragment was released by EcoRI digestion.
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PMID:Cloning and amplification of rabbit alpha- and beta-globin gene sequences into Escherichia coli plasmids. 32 53

We present a novel method for the PCR amplification of unknown DNA that flanks a known segment directly from human genomic DNA. PCR requires that primer annealing sites be present on each end of the DNA segment that is to be amplified. In this method, known DNA is placed on the uncharacterized side of the sequence of interest via DNA polymerase mediated generation of a PCR template that is shaped like a pan with a handle. Generation of this template permits specific amplification of the unknown sequence. Taq (DNA) polymerase was used to form the original template and to generate the PCR product. 2.2 kb of the beta-globin gene, and 657 bp of the 5' flanking region of the cystic fibrosis transmembrane conductance regulator gene, were amplified directly from human genomic DNA using primers that initially flank only one side of the region amplified. This method will provide a powerful tool for acquiring DNA sequence information.
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PMID:Sequence specific generation of a DNA panhandle permits PCR amplification of unknown flanking DNA. 137 52

A novel beta-chain, beta 126(H4)Val----Gly, electrophoretically silent, was detected by reverse-phase high performance liquid chromatography in three unrelated families from Naples (Southern Italy) and accounted for about 30% of the total beta-chains. The amino acid substitution was detected by HPLC fingerprint. The eight heterozygous patients showed hematologic and biosynthetic alterations of mild beta-thalassemia type. The hemoglobin variant showed abnormal stability features. It was unstable in the heat stability and isopropanol precipitation tests, but did not cause a hemolytic syndrome in vivo and was stable in a time-course experiment of biosynthesis in vitro. DNA polymerase chain reaction direct sequencing of the mutated gene from 135 nt upstream of the cap site to 106 nt downstream of the polyadenylation site showed only the beta 126 GTG----GGG mutation, which was confirmed in the other patients by allele-specific oligonucleotide hybridization. The mutation was found to be associated with a type II beta-globin framework and restriction fragment length polymorphism haplotype V. The novel variant was named hemoglobin Neapolis.
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PMID:Hemoglobin Neapolis, beta 126(H4)Val----Gly: a novel beta-chain variant associated with a mild beta-thalassemia phenotype and displaying anomalous stability features. 195 92

This paper describes DNA polymerase chain reaction (PCR) amplification directly from dried blood specimens for the detection of the beta-thalassemia mutation in China. Target DNA was amplified to span the beta-globin gene regions, which included ten types of mutation sites specific for Chinese beta-thalassemias. Ten kinds of oligonucleotide probes were constructed and used to hybridize with the amplified DNA. A total of 170 beta-thalassemia alleles originating from eastern, southwestern and southern China were analyzed. The results revealed that the distributions of different types of mutations were different in the three regions. The most common types in southern China were a frameshift at codons 41/42 and a C----T substitution at IVS II n.654, the most frequent types in southwestern China were codon 17 and IVS II n.654 mutations, and the predominant mutations in eastern China were frameshifts at codons 41/42 and 71/72.
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PMID:Detection of beta-thalassemia mutations in the Chinese using amplified DNA from dried blood specimens. 229 48

In an ongoing effort to identify point mutations causing beta-thalassaemia, we have found two previously unreported mutations which are located in the Poly A site of the beta-globin gene. The screening programme used amplified DNA and dot-blot hybridization with several 32P-labelled oligonucleotide probes. DNA samples which remained unidentified by this methodology were subjected to sequencing with 32P-labelled primers and modified T7 DNA polymerase. The newly discovered mutations were confirmed by the dot-blot hybridization technique. One type concerned an AATAAA----AATGAA mutation in the polyadenylation site and was found in one family from Yugoslavia (including one patient with the C----T mutation at codon 29 in trans), one from Bulgaria (the patient had the G----A mutation at IVS-I-110 in trans), and one from Greece (this patient had the C----G mutation at IVS-II-745 in trans). Haematological data for three simple heterozygotes suggested a rather mild beta(+)-thalassemia. The second type involved an AATAAA----AATAGA mutation and was found in one family from Malaysia. The propositus had the beta E mutation on the other chromosome, was originally diagnosed as mild Hb E-beta(+)-thalassaemia, and had Hb A and Hb E percentages which were nearly the same.
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PMID:Two novel polyadenylation mutations leading to beta(+)-thalassemia. 237 10

We report the characterization of a beta zero-thalassemia in an American Black with unusually high HbA2 and HbF levels. Genomic southern analysis indicated that the individual was heterozygous for a deletion that began within the second intervening sequence of the beta-globin gene and extended approximately 1.4 kb in the 5' direction. A clone spanning the breakpoint on the abnormal chromosome was isolated and further mapped, and the deletion joint was sequenced. Comparison of the normal beta-globin gene and its 5' flanking region with the deletion joint sequence indicated that the 5' breakpoint for this deletion was 484 base pairs (bp) 5' to the transcriptional start site for the beta-globin gene and the 3' breakpoint was 908 bp into the beta-globin gene; the deletion removed a total of 1,393 bp. Comparison of the normal 5' and 3' breakpoint sequences indicated that this deletion was the result of a "clean" nonhomologous breakage and reunion event; ie, no spurious bases were added during the recombinational event. Analysis of the breakpoints of this deletion together with the breakpoints of two other small deletions involving the beta-globin gene suggests that the breakpoints may occur at DNA polymerase alpha pause sites.
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PMID:Molecular characterization of a beta zero-thalassemia resulting from a 1.4 kilobase deletion. 245 98

The beta-globin genes from a Thai patient compound heterozygous for beta-thalassemia and HbE disease were investigated. The 3.0-kilobase fragment containing the entire beta-globin gene was amplified by polymerase chain reaction, using Taq DNA polymerase followed by direct cloning of the amplified product into plasmid DNA. Sequence analysis of the thalassemia gene revealed only one base change, a C-A transversion within codon for an amino acid 35. This new mutation creates a premature terminator, TAA, an ochre codon, and results in a beta 0-thalassemia phenotype. The same result was obtained when this mutation was analyzed using a conventional cloning technique, direct sequencing of the amplified product, and hybridization with allele-specific oligonucleotide probes. No misincorporation was detected in the sequence analysis of the 3.0-kilobase insert of five clones of the amplified products obtained from genomic DNA of a normal individual. This approach is a rapid and accurate method for molecular cloning of the beta-globin gene and also other genes, the partial nucleotide sequences of which are known.
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PMID:A novel ochre mutation in the beta-thalassemia gene of a Thai. Identification by direct cloning of the entire beta-globin gene amplified using polymerase chain reactions. 254 42

A modified assay of nick-translation of nuclei has been developed to study the chromatin structure of human beta-like globin genes in nuclei of K 562 cell line. Nuclei were gently digested with DNase I and nick-translated with E. coli DNA polymerase I in the presence of 32P-triphosphate nucleotides. The total DNA from the labelled nuclei was used as probes to hybridize restricted fragments of beta-like globin genes which have been immobilized on Diazobenzyloxymethyl (DBM) paper. Using this approach we have observed that in K 562 nuclei all beta-like globin genes, including epsilon, gamma, delta, and beta-globin genes and human 18 S ribosomal genes are preferentially labelled in comparison to alpha-lactalbumin and c-sis genes which do not express in K 562 cells, but the total DNA from nick-translated nuclei of a nonerythroid cell line hybridized none of those genes except for 18 S ribosomal gene. This assay is a simple and fast method for surveying chromatin structure of any individual DNA sequence in nuclei once the corresponding clone is available.
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PMID:A study of the chromatin structure of human beta-like hemoglobin genes in K562 cell line with a modified assay of nick-translation of nuclei. 262 96

DNA amplification combined with the use of synthetic oligonucleotide probes has become an important tool in the identification of base substitutions. We report the use of this DNA amplification technique for the detection of mutations in beta-thalassemia. A series of oligonucleotide primers are synthesized which span the beta-globin gene; one primer is complementary to the coding strand and the other to the non-coding strand. The primers are chosen so that there is little homology with other DNA segments, especially the delta gene. Each set of primers spans an area of the gene between 100 and 300 bp, while the suspected mutation point is located between these two primers. With the use of such a primer set, the beta-globin gene region is amplified by denaturation, annealing and DNA synthesis. The amplification cycle is repeated 25-30 times, using the Klenow fragment of DNA polymerase I. The resulting amplified DNA is hybridized with normal and synthetic deoxynucleotide probes using a standard dot-blot method. We have designed a set of primers and experimental conditions which should prove useful to diagnostic centers for detection of numerous beta-thalassemia mutations.
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PMID:The detection of beta-globin gene mutations in beta-thalassemia using oligonucleotide probes and amplified DNA. 282 12

One of the easiest and most sensitive methods of detecting mutations in the beta-globin gene leading to beta-thalassemia is by the use of oligonucleotide probes. The current method involves digestion of 5-10 micrograms of genomic DNA followed by gel electrophoresis, and blotting onto nitrocellulose. The membrane is then hybridized with a 32P-radiolabeled oligonucleotide probe containing the specific point mutation of interest. Finally, the membrane is subjected to X-ray film for 3-10 days. We wish to report a method for detecting these mutations which involves 1 microgram of genome DNA or less. The method involves the use of a gene amplification technique. A series of primers are synthesized which span the beta-globin gene. In each primer set, one primer is complementary to the beta-gene and the other primer is complementary to the non-coding strand. The suspected mutation point is located between these two primers. With the use of this primer set, the beta-globin gene region is amplified by denaturing, annealing, and DNA synthesis. The amplification cycle is repeated 25 to 30 times. The amplification is conducted using the Klenow fragment of DNA polymerase I or Taq polymerase in the presence of all four deoxynucleotide triphosphates. The resulting amplified DNA is applied to a nylon membrane with the aid of a dot-blot apparatus and directly hybridized with normal and mutant deoxynucleotide probes. The entire process requires one to two days. More than 300 beta-thalassemia homozygotes have been identified in our laboratories; over 20 different mutations have been observed.
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PMID:Newer developments in the identification of beta-thalassemia. 320 97


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