Calibration and Storage of DNA Competitors Used for Contamination-Protected Competitive PCR
DNA fragments used as standards in competitive PCR were precisely calibrated using HPLC and commercially available DNA molecular mass markers. The accuracy of calibration was reflected by data that differed by only 2% from the mean when two independently purified and calibrated competitor preparatio...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
1997-10-01
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| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/97234st07 |
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| _version_ | 1850152515220275200 |
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| author | Thomas Köhler Anne-Katrin Rost Harald Remke |
| author_facet | Thomas Köhler Anne-Katrin Rost Harald Remke |
| author_sort | Thomas Köhler |
| collection | DOAJ |
| description | DNA fragments used as standards in competitive PCR were precisely calibrated using HPLC and commercially available DNA molecular mass markers. The accuracy of calibration was reflected by data that differed by only 2% from the mean when two independently purified and calibrated competitor preparations were compared. Highly dilute competitor solutions were stable at -20°C for up to 1 year in the presence of carrier HindIII-digested γDNA, but progressive loss of competitor DNA with increasing storage time was observed when carrier DNA was omitted from the solution. Applying 0.2 U uracil-DNA glycosylase (UDG) per assay of remaining temperaturestable activity did not effect the ratios of synthesized products. This study describes quality management in PCR quantitation that is useful for the measurement of multidrug resistance-associated protein (MRP) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene transcripts. |
| format | Article |
| id | doaj-art-0986ea9ca53340fcb73f34c6a38b399b |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 1997-10-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-0986ea9ca53340fcb73f34c6a38b399b2025-08-20T02:25:58ZengTaylor & Francis GroupBioTechniques0736-62051940-98181997-10-0123472272610.2144/97234st07Calibration and Storage of DNA Competitors Used for Contamination-Protected Competitive PCRThomas Köhler0Anne-Katrin Rost1Harald Remke21University of Leipzig, Leipzig, Germany1University of Leipzig, Leipzig, Germany1University of Leipzig, Leipzig, GermanyDNA fragments used as standards in competitive PCR were precisely calibrated using HPLC and commercially available DNA molecular mass markers. The accuracy of calibration was reflected by data that differed by only 2% from the mean when two independently purified and calibrated competitor preparations were compared. Highly dilute competitor solutions were stable at -20°C for up to 1 year in the presence of carrier HindIII-digested γDNA, but progressive loss of competitor DNA with increasing storage time was observed when carrier DNA was omitted from the solution. Applying 0.2 U uracil-DNA glycosylase (UDG) per assay of remaining temperaturestable activity did not effect the ratios of synthesized products. This study describes quality management in PCR quantitation that is useful for the measurement of multidrug resistance-associated protein (MRP) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene transcripts.https://www.future-science.com/doi/10.2144/97234st07 |
| spellingShingle | Thomas Köhler Anne-Katrin Rost Harald Remke Calibration and Storage of DNA Competitors Used for Contamination-Protected Competitive PCR BioTechniques |
| title | Calibration and Storage of DNA Competitors Used for Contamination-Protected Competitive PCR |
| title_full | Calibration and Storage of DNA Competitors Used for Contamination-Protected Competitive PCR |
| title_fullStr | Calibration and Storage of DNA Competitors Used for Contamination-Protected Competitive PCR |
| title_full_unstemmed | Calibration and Storage of DNA Competitors Used for Contamination-Protected Competitive PCR |
| title_short | Calibration and Storage of DNA Competitors Used for Contamination-Protected Competitive PCR |
| title_sort | calibration and storage of dna competitors used for contamination protected competitive pcr |
| url | https://www.future-science.com/doi/10.2144/97234st07 |
| work_keys_str_mv | AT thomaskohler calibrationandstorageofdnacompetitorsusedforcontaminationprotectedcompetitivepcr AT annekatrinrost calibrationandstorageofdnacompetitorsusedforcontaminationprotectedcompetitivepcr AT haraldremke calibrationandstorageofdnacompetitorsusedforcontaminationprotectedcompetitivepcr |