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)

Achieving the capacity to detect minimal numbers of neoplastic cells is a major cancer diagnostic challenge. Chromosomal translocations such as the t(14;18)(q32;q21) found in follicular and some nonfollicular lymphomas provide a tumor-specific molecular marker. The 14;18 breakpoints are focused at one of six immunoglobulin heavy chain joining (JH) regions on chromosome 14 and a small major breakpoint region (MBR) of the BCL2 gene on chromosome 18. We utilized universal oligonucleotide primers of a region 5' to the BCL2 MBR and at the 3' end of JH segments to initiate a DNA polymerase chain reaction that amplified these BCL2-JH junctures. Use of thermostable DNA polymerase enabled annealing and synthesis steps at temperatures approaching the melting point of the primers, providing a sensitive and specific assay capable of detecting 1 lymphoma cell in 10(6) normal cells. This technique identified the subclinical presence of leukemic cells in all seven patients examined, including two in clinical remission. It also assessed the effectiveness of protocols designed to purge malignant cells from marrow. Moreover, this approach enabled the rapid DNA sequencing of chromosomal breakpoints without their molecular cloning. This assay markedly refines the capacity to detect minimal residual disease and should improve the ability to determine the stage of disease, stratify treatment, and evaluate therapy.
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PMID:Thermostable DNA polymerase chain amplification of t(14;18) chromosome breakpoints and detection of minimal residual disease. 313 63

Anaplastic large cell lymphoma (ALCL) is a distinct clinicopathologic variant of intermediate grade non-Hodgkin's lymphomas (NHL) composed of large pleomorphic cells that usually express the CD30 antigen and interleukin (IL)-2 receptors, and is characterized by frequent cutaneous and extranodal involvement. With variable frequency ALCL bear the t(2;5)(p23;q35) chromosomal translocation that fuses the nucleophosmin (NPM) gene on chromosome 5q35 to a novel protein kinase gene, Anaplastic Lymphoma Kinase (ALK), on chromosome 2p23. We determined the frequency of this translocation with a novel DNA polymerase chain reaction (PCR) technique using 0.5 microgram of genomic DNA, 5'-primers derived from the NPM gene and 3'-primers derived from the ALK gene and hybridization with internal probes. The presence of amplifiable DNA in the samples was tested with the inclusion in the PCR reaction of oligonucleotide primers designed to amplify a 3016-bp fragment from the beta-globin locus. NMP-ALK fusion amplicons were detected using DNA isolated either from all three ALCL cell lines tested, or from all four primary ALCL tumors known to contain the t(2;5)(p23;q35) translocation. Nested amplicons were detected by hybridization in 100% of specimens diluted 10(4)-fold and in 20% of those diluted 10(5)-fold. We subsequently examined archival genomic DNA from 20 patients with ALCL, 39 with diffuse large cell, 2 with mantle cell, 20 with peripheral T cell, 13 with low-grade NHL, 31 with Hodgkin's disease (HD), and 6 with lymphomatoid papulosis. Fusion of the NPM and ALK genes was detected in three of 18 patients with ALCL who had amplifiable DNA (17%, 95% confidence intervals 4% to 41%), but not in any patients with other NHL, HD, or lymphomatoid papulosis. The amplicon sizes were different in all cell lines and patients reflecting unique genomic DNA breakpoints. We conclude that with genomic DNA-PCR the rearrangement of the NPM and ALK loci is restricted to patients with ALCL. Further studies are needed to determine the prognostic significance of the NPM-ALK rearrangement, to determine whether its detection can aid in the differential diagnosis between ALCL. Hodgkin's disease, and lymphomatoid papulosis, and to establish the usefulness of the genomic DNA PCR in the monitoring of minimal residual disease in those patients whose tumors bear the t(2;5).
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PMID:Amplification of genomic DNA demonstrates the presence of the t(2;5) (p23;q35) in anaplastic large cell lymphoma, but not in other non-Hodgkin's lymphomas, Hodgkin's disease, or lymphomatoid papulosis. 926 95

Anaplastic large cell lymphoma (ALCL) is an intermediate grade Non-Hodgkin's lymphoma (NHL) characterized by the frequent presence of the t(2;5)(p23;q35). This translocation fuses the nucleophosmin (NPM) gene on chromosome 5q35 to a protein kinase gene (Anaplastic Lymphoma Kinase, ALK) on chromosome 2p23. In order to determine the frequency of t(2;5) we used a DNA polymerase chain reaction (PCR) amplification using genomic DNA, 5'-primers derived from the NPM gene, and 3'-primers derived from the ALK gene. The presence of amplifiable DNA in the samples was established with PCR and oligonucleotide primers designed to amplify a 3,016 bp fragment from the beta-globin locus. The t(2;5) PCR assay was established using DNA isolated from three t(2;5)-positive ALCL cell lines. Its ability to amplify genomic DNA prepared for routine molecular diagnostic use was validated using archival DNA from four ALCL tumors known to be t(2;5)-positive. Its sensitivity was established by serially diluting t(2;5)-positive DNA in normal DNA: amplicons were generated in 100% of reactions diluted 10(4)-fold (6-8 cells per tube) and in 30% of those diluted 10(5)-fold (0.6-0.8 cells per tube.) We subsequently analyzed archival genomic DNA extracted from 38 ALCL, 77 NHLs, 37 Hodgkin's lymphomas, and 9 lymphomatoid papuloses. The t(2;5) was detected in 6 ALCLs (16%, 95% confidence intervals 6%-31%), but not in any other lymphoma, or in lymphomatoid papulosis. By using the published sequence of the fourth NPM intron that is involved in t(2;5) and by sequencing the individual tumor amplicons and also the normal ALK intron that is involved in t(2;5), we established that all breakpoints involve the same introns in the ALK and NPM loci. Detailed analysis demonstrated that each translocation generates a unique breakpoint sequence, and suggested that sequence homology between the ALK and NPM intron sequences may be involved in the translocation. We conclude that genomic DNA-PCR is useful for the detection of t(2;5) that in our patient population is restricted to ALCL and is not detectable in other NHL, Hodgkin's disease, or lymphomatoid papulosis. More work is needed to determine the prognostic significance of t(2;5), and to establish the utility of the genomic DNA PCR in monitoring minimal residual disease.
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PMID:Genomic DNA amplification and the detection of t(2;5)(p23;q35) in lymphoid neoplasms. 964 64

To measure mutation load or to detect minimal residual disease, a robust method for identifying one mutant allele in the range of 10(6)-10(9) wild-type alleles would be advantageous. Herein, we present evidence that pyrophosphorolysis-activated polymerization (PAP) has the potential to provide a highly specific and robust method of allele-specific amplification if DNA polymerases with higher pyrophosphorolysis activity can be found or engineered. In PAP, pyrophosphorolysis and polymerization by DNA polymerase are coupled serially by utilizing a pyrophosphorolysis-activatable oligonucleotide (P*). P*, which is an allele-specific oligonucleotide with a dideoxynucleotide at the 3' terminus, can be activated by pyrophosphorolysis to remove the 3' terminal dideoxynucleotide in the presence of pyrophosphate (PPi) and the complementary strand of the allelic template; then the activated P* can be extended by DNA polymerization. Specificity results from both pyrophosphorolysis and polymerization because significant nonspecific amplification requires the combination of mismatch pyrophosphorolysis and misincorporation by the DNA polymerase, which is an extremely rare event. Proof of principle has been achieved with a polymorphic site within the human D1 dopamine receptor gene. The effects of the dideoxyoligonucleotide sequences, DNA polymerases, PPi concentrations, allele-specific templates, pH and dNTP concentrations were examined.
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PMID:Pyrophosphorolysis-activated polymerization (PAP): application to allele-specific amplification. 1108 70

We find that Type II DNA polymerases can catalyze pyrophosphorolysis, the reverse reaction of DNA polymerization. This property is applied utilizing pyrophosphorolysis-activated polymerization (PAP), a method of nucleic acid amplification using serial coupling of pyrophosphorolysis and polymerization. PAP can be used for ultrarare allele detection (detection of minimal residual disease and cancer risk assessment through measurement of mutation load) and for microarray-based scanning for unknown mutations. Herein, we show that Type II DNA polymerases efficiently catalyze template-dependent pyrophosphorolysis to activate oligonucleotides blocked at their 3' termini with acyclonucleotides in which a 2-hydroxyethoxymethyl group substitutes for the 2'-deoxyribofuranosyl sugar. Type II archeon DNA polymerases Vent (exo-) and Pfu (exo-) can be utilized for PAP or a bidirectional form of PAP with acyclonucleotide-blocked oligonucleotides, but not with dideoxynucleotide-blocked oligonucleotides. In contrast, a Type I DNA polymerase, TaqFS, can utilize either acyclonucleotide-blocked or dideoxynucleotide-blocked oligonucleotides. These findings expand the potential of nascent PAP technology.
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PMID:Pyrophosphorolysis by Type II DNA polymerases: implications for pyrophosphorolysis-activated polymerization. 1465 41

The need for detection of minority mutations (i.e., a few mutants within a high excess of wild-type alleles) arises frequently in the field of cancer and molecular genetics. Current mutation detection technologies are limited by several technical factors when it comes to the detection of minority point mutations, including generation of misincorporations by the DNA polymerase during PCR amplification. Primer ligation-mediated PCR methodologies for detection of mutations in an excess wild-type sequences are described, that can be applied for detection of both known and unknown minority point mutations. Furthermore, a new methodology is described, hairpin-PCR, which has the potential to completely eliminate PCR errors from amplified sequences, prior to minority mutation detection. Combination of these technologies can effectively tackle the problem of minority mutation detection, in order to pursue demanding applications such as identification of cancer cells at an early stage, detection of mutations in single cells, identification of minimal residual disease, or investigation of mechanisms of spontaneous mutagenesis.
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PMID:PCR-based detection of minority point mutations. 1510 70