Routinely Prepared Cells for Cytogenetic Analysis Stored at −20 °C for Several Years can be Used for RT-PCR-Based Detection of Chromosomal Aberrations in Leukemias
Philippe SchafhausenRobert SchochMaike NickelsenTorsten HaferlachStefan JenischK Weber-MatthiesenBrigitte SchlegelbergerMartin KrönkeW. GaßmannH. Löffler
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Primer (cosmetics)
Chronic myelogenous leukemia
The comparison of K m , and V max values for various primers in the reaction of polymerization catalyzed by the human immunodeficiency virus type‐1 (HIV‐1) reverse transcriptase was carried out. The primers were: (a) complementary to the template, (b) partially complementary with mismatched nucleotides at different positions from the 3′ end or (c) non‐complementary. Non‐complementary primers were not elongated by HIV‐1 reverse transcriptase. However, if they contained only one residue complementary to the template or an abasic unit at the 3′ end, they could serve as primers. The most effective discrimination between matched and mismatched primers, due to a decrease in the affinity and V max , was found in the case of oligonucleotides containing non‐complementary bases at the second or third position from the 3′ end of the primer. The efficiency of discrimination by HIV‐1 reverse transcriptase between matched and mismatched base‐paired primers was about 1–1.5 orders of magnitude lower than that of procaryotic, eucaryotic and archaebacterial DNA polymerases and avian myeloblastosis virus reverse transcriptase. Oligonucleotides such as (dT) 4 (dCdG) k (dT) 4 showed higher affinity for the enzyme than (dT) 4 or (dT) 8 primers. These data suggest that HIV‐1 reverse transcriptase, in contrast to procaryotic, eucaryotic and archaebacterial DNA polymerases, forms additional contacts with the 5′‐end region of the non‐complementary primer. In addition, using tRNA 3 Lys , the natural primer of HIV‐1, it was shown that the p66 subunit of reverse transcriptase can be crosslinked, in the presence of a platinum derivative, to the 5′ end of tRNA. Thus. besides the normal binding site for the 3′ end of tRNA, which is crucial for the initiation of cDNA synthesis, the 5′ end of the tRNA also interacts with a specific site on the enzyme.
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RNA-Directed DNA Polymerase
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More than five different primer pairs have been used for the detection of Mycobacterium tuberculosis deoxyribonucleic acid (DNA) with the polymerase chain reaction (PCR).The sensitivity and specificity of PCR were evaluated using three different primer pairs in the detection of M. tuberculosis in paraffin-embedded tissues.Thirty-eight tissue specimens from 23 patients were studied. Eighteen samples were obtained from 10 tuberculosis patients, and 20 samples obtained from 13 patients with other diseases were used as negative controls. DNA extracted from paraffin-embedded tissues was used directly for PCR amplification using primers IS1 and IS2 to amplify a 123 base pair (bp) region of IS6110, sjMT3 and sjMTr2 to amplify a 281 bp region of protein antigen b, and INS1 and INS2 to amplify a 245 bp region of IS986. Each amplification was performed double-blinded and repeated three times including positive and negative control samples.IS1 and IS2 gave a positive result in each of the double samples obtained from eight tuberculosis patients and in the single samples obtained in the two others, sjMT3 and sjMTr2 detected 13 of the 18 tuberculosis samples, and INS1 and INS2 detected only three of the 18.These results highlight the importance of selecting appropriate primers to obtain high sensitivity in detecting M. tuberculosis in paraffin-embedded tissues by PCR.
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Low copy number
Mycobacterium tuberculosis complex
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Two primer sets were chosen for the detection of Haemophilus influenzae in cerebrospinal fluid by polymerase chain reaction (PCR) DNA amplification. One primer set was selected from sequences encoding a capsulation-associated protein and reacted with target DNA from all 15 capsulate H. influenzae strains (all serotypes) examined. The other primer set was selected from the DNA sequence of a gene encoding for outer-membrane protein P6 and reacted with the 15 capsulate and 10 non-capsulate strains of H. influenzae tested. This primer set also reacted with the closely related species H. haemolyticus and H. aegyptius, and with two of nine H. parainfluenzae strains. In reconstruction experiments, PCR DNA amplification was able to detect as few as five H. influenzae cells when 40 cycles of amplification were used. Two hundred cerebrospinal fluid (CSF) samples collected consecutively from patients suffering from meningitis were investigated by PCR; 40 were culture-positive for H. influenzae and 39 of these were also clearly positive in the PCR test with both primer sets. Contamination occurred to some extent with 40 cycles of amplification but was completely eliminated when the number of cycles was reduced to 35. We conclude that the two primer sets are appropriate for the detection of H. influenzae by PCR, each having its own specificity. When these two primer sets are used, PCR is a technique of equivalent sensitivity to culture for the detection of H. influenzae in CSF.
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Nested polymerase chain reaction (nested PCR) was used to separately amplify part of gag, pol, and env genes of human immunodeficiency virus type 1 (HIV-1) to evaluate that primer specific to either gag (SK380/390&SK38/39), pol (JA17/18&JA19/20), or env (JA9/10&JA11/12) genes is suitable for HIV-1 PCR based diagnosis in Thailand. The positive PCR results in 70 HIV-1 infected adults are 100, 97, 89 per cent and in 75 HIV-1 infected infants are 100, 94, 74 per cent by gag, pol, env primer, respectively. The specificity of all three primer sets is 100 per cent. The unamplified samples by pol and env primers were identified as HIV-1 subtype E by PELISA method. False negative in HIV-1 PCR based diagnosis caused by high genetic variation of HIV-1 can be overcome by using several primer sets as shown in this study.
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Group-specific antigen
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The skill of PCR primer design with software is introduced in this paper.Based on the principle of PCR-primer design,the usage of two kinds of popular primer-design software was reviewed detailedly,including their merit,demerit and usage skills.It is recommended to use Premier Primer 5 for the usual automatic primers search,but Oligo 6 primers analysis.
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Primer dimer
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Rhesus macaque
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Nonnucleoside reverse transcriptase inhibitors (NNRTI) are a group of small hydrophobic compounds with diverse structures that specifically inhibit HIV-1 reverse transcriptase (RT). NNRTIs interact with HIV-1 RT by binding to a single site on the p66 subunit of the p66 / p51 heterodimeric enzyme, termed the NNRTI-binding pocket (NNRTI-BP). This binding interaction results in both short-range and long-range distortions of RT structure. In this article, we review the structural, computational and experimental evidence of the NNRTI-induced conformational changes in HIV-1 RT and relate them to the mechanism by which these compounds inhibit HIV-1 reverse transcription.
RNase H
Reverse-transcriptase inhibitor
RNA-Directed DNA Polymerase
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The sealing effects of a number of primer sealers, such as cationic primer, waterborne epoxy primer, silica sol modified primer, and conventional acrylic primer, are discussed theoretically and compared experimentally, in terms of air permeability, adhesion, mechanical properties, alkali and water resistances.
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Cationic polymerization
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Primer binding site
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Objective: To investigate the diagnosis of atypical chronic myelogenous leukemia.Methods: Through the comparison between atypical chronic myelogenous leukemia (12 cases) and typical chronic myelogenous leukemia (23 cases), we can find out that atypical chronic myelogenous leukemia was different from typical chronic myelogenous leukemia in clinical characteristics and morphology features.Results:First of all,the patients' age is always old.In most cases, there was not anemia but platelet was slightly higher than the normal, swelling of spleen was not unconspicuous, and the neutrophil alkaline phosphatase score was normal or slightly decreased.Philadelphia (Ph) chromosome was undetectable by polymerase chain reaction (PCR) as well.Conclusion: To diagnose this disease, the course of disease must be observed in succession which is aggravated along with the time passing and the other therapies are invalid.
Chronic myelogenous leukemia
Philadelphia chromosome
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