High-Temperature, Nonradioactive Primer Extension Assay for Determination of a Transcription-Initiation Site
We have developed a simple and safe method for the determination of a transcription-initiation site. In this method, reverse transcriptase of the avian myeloblastosis virus or rTth DNA polymerase from Thermus thermopilus was used with a fluorescein isothiocyanate (FITC)-labeled primer. The primer-ex...
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| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
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Taylor & Francis Group
1998-07-01
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| Series: | BioTechniques |
| Online Access: | https://www.future-science.com/doi/10.2144/98251st02 |
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| author | M. Yamada H. Izu T. Nitta K. Kurihara T. Sakurai |
| author_facet | M. Yamada H. Izu T. Nitta K. Kurihara T. Sakurai |
| author_sort | M. Yamada |
| collection | DOAJ |
| description | We have developed a simple and safe method for the determination of a transcription-initiation site. In this method, reverse transcriptase of the avian myeloblastosis virus or rTth DNA polymerase from Thermus thermopilus was used with a fluorescein isothiocyanate (FITC)-labeled primer. The primer-extension reaction can be performed at a high temperature, which reduces the hindering effect of the secondary structure in RNA, and can omit the annealing step between RNA and the primer. Almost all steps can be done in one tube. This procedure can provide reliable and reproducible data when compared with the conventional procedure at low temperature. Moreover, the sequencing ladder that is required for determining the position of extended products can be obtained with the same FITC-labeled primer. |
| format | Article |
| id | doaj-art-a8638d8bde904c2e8ade9b7b79fd3fe3 |
| institution | OA Journals |
| issn | 0736-6205 1940-9818 |
| language | English |
| publishDate | 1998-07-01 |
| publisher | Taylor & Francis Group |
| record_format | Article |
| series | BioTechniques |
| spelling | doaj-art-a8638d8bde904c2e8ade9b7b79fd3fe32025-08-20T02:26:00ZengTaylor & Francis GroupBioTechniques0736-62051940-98181998-07-01251727810.2144/98251st02High-Temperature, Nonradioactive Primer Extension Assay for Determination of a Transcription-Initiation SiteM. Yamada0H. Izu1T. Nitta2K. Kurihara3T. Sakurai41Yamaguchi University, Yamaguchi, Tokyo, Japan1Yamaguchi University, Yamaguchi, Tokyo, Japan1Yamaguchi University, Yamaguchi, Tokyo, Japan1Yamaguchi University, Yamaguchi, Tokyo, Japan1Yamaguchi University, Yamaguchi, Tokyo, JapanWe have developed a simple and safe method for the determination of a transcription-initiation site. In this method, reverse transcriptase of the avian myeloblastosis virus or rTth DNA polymerase from Thermus thermopilus was used with a fluorescein isothiocyanate (FITC)-labeled primer. The primer-extension reaction can be performed at a high temperature, which reduces the hindering effect of the secondary structure in RNA, and can omit the annealing step between RNA and the primer. Almost all steps can be done in one tube. This procedure can provide reliable and reproducible data when compared with the conventional procedure at low temperature. Moreover, the sequencing ladder that is required for determining the position of extended products can be obtained with the same FITC-labeled primer.https://www.future-science.com/doi/10.2144/98251st02 |
| spellingShingle | M. Yamada H. Izu T. Nitta K. Kurihara T. Sakurai High-Temperature, Nonradioactive Primer Extension Assay for Determination of a Transcription-Initiation Site BioTechniques |
| title | High-Temperature, Nonradioactive Primer Extension Assay for Determination of a Transcription-Initiation Site |
| title_full | High-Temperature, Nonradioactive Primer Extension Assay for Determination of a Transcription-Initiation Site |
| title_fullStr | High-Temperature, Nonradioactive Primer Extension Assay for Determination of a Transcription-Initiation Site |
| title_full_unstemmed | High-Temperature, Nonradioactive Primer Extension Assay for Determination of a Transcription-Initiation Site |
| title_short | High-Temperature, Nonradioactive Primer Extension Assay for Determination of a Transcription-Initiation Site |
| title_sort | high temperature nonradioactive primer extension assay for determination of a transcription initiation site |
| url | https://www.future-science.com/doi/10.2144/98251st02 |
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