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)

We have developed a simple and rapid nonradioactive method for detecting genetic variation and have applied it to the diagnosis of sickle cell anemia and beta-thalassemia. The procedure involves the selective amplification of a segment of the human beta-globin gene with oligonucleotide primers and a thermostable DNA polymerase, followed by hybridization of the amplified DNA with allele-specific oligonucleotide probes covalently labeled with horseradish peroxidase. The hybridized probes were detected with a simple colorimetric assay. We demonstrated the usefulness of this method in a retrospective analysis of two pregnancies at risk for beta-thalassemia and one at risk for sickle cell anemia, as well as in an analysis of nine DNA samples simulating three family sets.
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PMID:Diagnosis of sickle cell anemia and beta-thalassemia with enzymatically amplified DNA and nonradioactive allele-specific oligonucleotide probes. 340 66

A method is described for directly cloning enzymatically amplified segments of genomic DNA into an M13 vector for sequence analysis. A 110-base pair fragment of the human beta-globin gene and a 242-base pair fragment of the human leukocyte antigen DQ alpha locus were amplified by the polymerase chain reaction method, a procedure based on repeated cycles of denaturation, primer annealing, and extension by DNA polymerase I. Oligonucleotide primers with restriction endonuclease sites added to their 5' ends were used to facilitate the cloning of the amplified DNA. The analysis of cloned products allowed the quantitative evaluation of the amplification method's specificity and fidelity. Given the low frequency of sequence errors observed, this approach promises to be a rapid method for obtaining reliable genomic sequences from nanogram amounts of DNA.
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PMID:Direct cloning and sequence analysis of enzymatically amplified genomic sequences. 346 61

We developed an experimental system to examine the effects of mutations in the adenovirus major late promoter in its correct genomic location during a productive infection. A virus was constructed whose genome could be digested to give a rightward terminal DNA fragment extending from the XhoI site at 22.9 map units, which can be ligated or recombined with plasmid DNA containing adenovirus sequences extending from 0 to 22.9 or 26.5 map units, respectively. Mutations were made by bisulfite mutagenesis in the region between base pairs -52 and -12 with respect to the cap site at +1 and transferred to the appropriate plasmids for viral reconstruction. Of 19 mutant plasmid sequences containing single or multiple G-to-A transitions, 14 could be placed in the viral genome with no apparent change in phenotype. These mutant sequences included those which contained four transitions in the string of G residues immediately downstream of the TATA box. There were no alterations in rates of transcription from the major late promoter, sites of transcription initiation, or steady-state levels of late mRNAs. All of the five mutant sequences which could not be placed in virus contained multiple transitions both up- and downstream of the TATA box. Two of these apparently lethal mutant sequences were used in promoter fusion experiments to test their ability to promote transcription of rabbit beta-globin sequences placed in the dispensable E1 region of the virus. Both sequences showed diminished ability compared with wild-type sequences to promote transcription in this context. Comparisons between these two sequences and the viable mutant sequences suggest a role for the string of G residues located between -38 and -33 in promoting transcription from the major late promoter. The data as a whole also demonstrate that the specific nucleotide sequence of this region of the major late promoter, which overlaps transcription elements of the divergent IVa2 transcription unit and coding sequences of the adenovirus DNA polymerase, is not rigidly constrained but can mutate extensively without loss of these several functions.
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PMID:Mutations in the adenovirus major late promoter: effects on viability and transcription during infection. 356 9

Direct sequencing of specific regions of genomic DNA became feasible with the invention of the polymerase chain reaction (PCR) which permits amplification of specific regions of DNA. Recently, human mitochondrial DNA was amplified and directly sequenced. Using a thermostable DNA polymerase of T. aquaticus (Saiki, R.K. et al., manuscript in preparation) in the PCR, we have applied a combination of PCR and direct sequence analysis of the amplified product to a human single-copy gene. We studied the genomic DNA of five patients with beta-thalassaemia whose mutant alleles were uncharacterized, and found two previously undescribed mutations, along with three known alleles. One new allele is a frameshift at codons 106-107 and the other is an A-C transversion at the cap site (+1) of the beta-globin gene. This latter is the first natural mutation observed at the cap site and it occurs in a gene which is poorly expressed.
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PMID:Characterization of beta-thalassaemia mutations using direct genomic sequencing of amplified single copy DNA. 368 54

A 4.4-kb PstI fragment containing the entire beta-gene of the human beta-globin gene cluster plus both 5'- and 3'-flanking sequences was used as a probe to study the chromosomal localization of the beta-gene by in situ hybridization. Using random oligonucleotides as primers, the beta-gene DNA was 3H-labeled with the large fragment of DNA polymerase I (Klenow fragment) to a specific activity of 1.2 X 10(8) cpm/micrograms. Almost 80% of hybridization grains observed were located on the distal short arm of chromosome 11. High-resolution chromosome analysis suggests a more precise location of the beta-gene to region 11p15.4----p15.5.
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PMID:High-resolution chromosomal localization of the beta-gene of the human beta-globin gene complex by in situ hybridization. 405 91

The repair activity of Escherichia coli DNA polymerase I (Klenow fragment) was used to prepare nonadecanucleotide hybridization probes which were complementary either to the normal human beta-globin (beta A) or to the sickle cell human beta-globin (beta s) gene. Template-directed polymerization of highly radiolabeled alpha[32P]deoxyribonucleoside triphosphates (dNTPs) onto nonamer and decamer primers produced probes with specific activities ranging from 1.0 X 10(10) to 2.0 X 10(10) dpm/micrograms. The extremely high specific activities of these probes made it possible to detect the beta A and beta S single-copy gene sequences in as little as 1 microgram of total human genomic DNA as well as to discriminate between the homozygous and heterozygous states.
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PMID:Allele-specific hybridization using oligonucleotide probes of very high specific activity: discrimination of the human beta A- and beta S-globin genes. 636 93

A new method has been developed that permits the rapid amplification of unknown DNA flanking a known site so that one can walk into an uncharacterized region of DNA. This method eliminates the steps and sequence artifacts associated with cloning and permits genome walking into unclonable regions of DNA. In this method, human genomic DNA is restriction enzyme digested and then ligated to the 3' end of a 5'-phosphorylated oligonucleotide using a short bridging oligonucleotide using a short bridging oligonucleotide as a splint. The phosphorylated oligonucleotide is designed to create 5'-end extensions that are complementary to the known sequence. Following denaturation and reannealing under dilute conditions that promote intra-strand annealing and under high stringency, only those DNA strands that contain the known sequence will form a stem-loop structure with a recessed and phosphorylated 5' end. This stem-loop renders a substrate for a subsequent heat-stable ligation reaction to another oligonucleotide that anneals to the known sequence immediately adjacent to the phosphorylated oligonucleotide high-stringency annealing site. The oligonucleotide appended to the phosphorylated oligonucleotide by the heat-stable ligase can, when present in its free, non-ligated form, prime DNA polymerase-mediated amplification of those strands modified by site-specific ligation to this same oligonucleotide. This is followed by one or two nested DNA amplifications, with the final amplification primed by the phosphorylated oligonucleotide in its free, non-ligated form. We successfully applied this method to the specific amplification of 2.2 kb of DNA flanking the 5' end of the cystic fibrosis transmembrane conductance regulator cDNA using primers that anneal to the cDNA sequence and to the specific amplification of 2.2 kb of human genomic beta-globin DNA flanking the primer annealing sites.
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PMID:A method for the amplification of unknown flanking DNA: targeted inverted repeat amplification. 750 1

We have used the polymerase chain reaction (PCR) to amplify up to 22 kb of the beta-globin gene cluster from human genomic DNA and up to 42 kb from phaga lambda DNA. We have also amplified 91 human genomic inserts of 9-23 kb directly from recombinant lambda plaques. To do this, we increased pH, added glycerol and dimethyl sulfoxide, decreased denaturation times, increased extension times, and used a secondary thermostable DNA polymerase that possesses a 3'-to 5'-exonuclease, or "proofreading," activity. Our "long PCR" protocols maintain the specificity required for targets in genomic DNA by using lower levels of polymerase and temperature and salt conditions for specific primer annealing. The ability to amplify DNA sequences of 10-40 kb will bring the speed and simplicity of PCR to genomic mapping and sequencing and facilitate studies in molecular genetics.
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PMID:Effective amplification of long targets from cloned inserts and human genomic DNA. 820 50

Prenatal DNA-diagnosis of beta-thalassemia in a family from Azerbaijan revealed two mutations new for this region--G-A transition at codon 15 and G-C transversion at position 5 of the intron 1. Prenatal diagnosis was carried out by direct sequencing of in vitro amplified (PCR) beta-globin gene fragments with a modified Sanger technique using thermostable DNA polymerase. The absence of parents mutations in the fetal DNA allowed us to conclude that the fetus is normal. The diagnosis was proved at hematological testing of the baby borne.
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PMID:[Use of the polymerase chain reaction to detect beta-thalassemia mutations in heterozygous carriers from Azerbaijan while performing prenatal DNA-diagnosis]. 833 39

During the latent period of murine erythroleukemia (MEL) cell differentiation, c-myc levels showed a significant change and the overexpression of the transferred c-myc gene inhibited the commitment and differentiation of MEL cells, suggesting that c-Myc may be a key molecule for the commitment. Since c-Myc may function as a DNA binding transcription factor, we examined whether c-Myc regulates the latent period genes (hsp and hsc70, MER5, Id and Spi-1 genes) and the erythroid-specific genes [beta-globin, glycophorin, delta-aminolevulinic acid synthase (ALAS-E), GATA-1 and erythropoietin receptor (EpoR)] in the MEL cell transformant having transferred c-myc gene. The overexpression of c-myc gene affected the latent period genes in different ways: hsc and hsp 70 genes and Id gene were positively regulated, while expression of MER5 gene was repressed. While c-myc is thought to be involved in DNA replication, its overexpression showed no effect on the expression of proliferating cell specific nuclear antigen or DNA polymerase a. The overexpression of c-myc repressed the expression of glycophorin, ALAS-E and beta-globin genes, of the five erythroid-specific genes, but had no effect on expression of GATA-1 or EpoR gene. These results suggest that c-Myc differentially regulates the expression of the latent period and erythroid-specific genes.
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PMID:c-Myc selectively regulates the latent period and erythroid-specific genes in murine erythroleukemia cell differentiation. 840 52


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