Potential influence of the first PCR cycles in real-time comparative gene quantifications
There is an underlying assumption in real-time PCR that the amplification efficiency is equal from the first cycles until a signal can be detected. In this study, we evaluated this assumption by analyzing genes with known gene copy number using real-time PCR comparative gene quantifications. Listeri...
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
2004-08-01
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| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/04372RR01 |
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| _version_ | 1850152167051100160 |
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| author | Hege Karin Nogva Knut Rudi |
| author_facet | Hege Karin Nogva Knut Rudi |
| author_sort | Hege Karin Nogva |
| collection | DOAJ |
| description | There is an underlying assumption in real-time PCR that the amplification efficiency is equal from the first cycles until a signal can be detected. In this study, we evaluated this assumption by analyzing genes with known gene copy number using real-time PCR comparative gene quantifications. Listeria monocytogenes has six 23S rRNA gene copies and one copy of the hlyA gene. We determined 23S rRNA gene copy numbers between 0.9 and 1.6 relative to hlyA when applying the comparative gene quantification approach. This paper focuses on the first cycles of PCR to explain the difference between known and determined gene copy numbers. Both theoretical and experimental evaluations were done. There are three different products (types 1–3) dominating in the first cycles. Type 1 is the original target, type 2 are undefined long products, while type 3 are products that accumulate during PCR. We evaluated the effects of type 1 and 2 products during the first cycles by cutting the target DNA with a restriction enzyme that cuts outside the boundaries of the PCR products. The digestion resulted in a presumed increased amplification efficiency for type 1 and 2 products. Differences in the amplification efficiencies between type 1, 2, and 3 products may explain part of the error in the gene copy number determinations using real-time PCR comparative gene quantifications. Future applications of real-time PCR quantifications should account for the effect of the first few PCR cycles on the conclusions drawn. |
| format | Article |
| id | doaj-art-c027751bd32a49c6ad3b8e5f0e97c14d |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 2004-08-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-c027751bd32a49c6ad3b8e5f0e97c14d2025-08-20T02:26:03ZengTaylor & Francis GroupBioTechniques0736-62051940-98182004-08-0137224625310.2144/04372RR01Potential influence of the first PCR cycles in real-time comparative gene quantificationsHege Karin Nogva0Knut Rudi11Norwegian Food Research Institute, ÅS, Norway1Norwegian Food Research Institute, ÅS, NorwayThere is an underlying assumption in real-time PCR that the amplification efficiency is equal from the first cycles until a signal can be detected. In this study, we evaluated this assumption by analyzing genes with known gene copy number using real-time PCR comparative gene quantifications. Listeria monocytogenes has six 23S rRNA gene copies and one copy of the hlyA gene. We determined 23S rRNA gene copy numbers between 0.9 and 1.6 relative to hlyA when applying the comparative gene quantification approach. This paper focuses on the first cycles of PCR to explain the difference between known and determined gene copy numbers. Both theoretical and experimental evaluations were done. There are three different products (types 1–3) dominating in the first cycles. Type 1 is the original target, type 2 are undefined long products, while type 3 are products that accumulate during PCR. We evaluated the effects of type 1 and 2 products during the first cycles by cutting the target DNA with a restriction enzyme that cuts outside the boundaries of the PCR products. The digestion resulted in a presumed increased amplification efficiency for type 1 and 2 products. Differences in the amplification efficiencies between type 1, 2, and 3 products may explain part of the error in the gene copy number determinations using real-time PCR comparative gene quantifications. Future applications of real-time PCR quantifications should account for the effect of the first few PCR cycles on the conclusions drawn.https://www.future-science.com/doi/10.2144/04372RR01 |
| spellingShingle | Hege Karin Nogva Knut Rudi Potential influence of the first PCR cycles in real-time comparative gene quantifications BioTechniques |
| title | Potential influence of the first PCR cycles in real-time comparative gene quantifications |
| title_full | Potential influence of the first PCR cycles in real-time comparative gene quantifications |
| title_fullStr | Potential influence of the first PCR cycles in real-time comparative gene quantifications |
| title_full_unstemmed | Potential influence of the first PCR cycles in real-time comparative gene quantifications |
| title_short | Potential influence of the first PCR cycles in real-time comparative gene quantifications |
| title_sort | potential influence of the first pcr cycles in real time comparative gene quantifications |
| url | https://www.future-science.com/doi/10.2144/04372RR01 |
| work_keys_str_mv | AT hegekarinnogva potentialinfluenceofthefirstpcrcyclesinrealtimecomparativegenequantifications AT knutrudi potentialinfluenceofthefirstpcrcyclesinrealtimecomparativegenequantifications |