Academic Journal
Single-Tube Method for Nucleic Acid Extraction, Amplification, Purification, and Sequencing
| العنوان: | Single-Tube Method for Nucleic Acid Extraction, Amplification, Purification, and Sequencing |
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| المؤلفون: | Margraf, Rebecca L, Page, Sam, Erali, Maria, Wittwer, Carl T |
| المصدر: | Clinical Chemistry ; volume 50, issue 10, page 1755-1761 ; ISSN 0009-9147 1530-8561 |
| بيانات النشر: | Oxford University Press (OUP) |
| سنة النشر: | 2004 |
| مصطلحات موضوعية: | Biochemistry (medical), Clinical Biochemistry |
| الوصف: | Background: The hepatitis C virus (HCV) genotype determines patient prognosis and duration of treatment, but sequencing of the gene is lengthy and labor-intensive. We used a commercially available nucleic acid extraction system to develop a single-tube extraction-to-sequencing (STETS) method for HCV genotyping. Methods: HCV RNA was purified and amplified in tubes coated with a solid-phase matrix that irreversibly bound nucleic acid during the extraction step. After reverse transcription-PCR, the amplicon was adsorbed to the original extraction matrix for purification and use in the subsequent sequencing reactions. Results: The STETS method generated genotyping-quality sequence for a range of HCV titers from 500 to 6 000 000 IU/mL. If a viral sample was detected during real-time reverse transcription-PCR, it could be sequenced and genotyped. Read lengths >600 bases were observed with the STETS method. Mixed infections were detected and genotyped if at least 15% of the minor species was present. Combining the STETS method with consecutive sequencing provided a means of performing both forward and reverse sequencing in a single tube. Conclusions: A single-tube nucleic acid extraction-to-sequencing method, which requires less time and labor than conventional methods, generates HCV sequence data that are equivalent to conventional methods and can be used to genotype HCV. |
| نوع الوثيقة: | article in journal/newspaper |
| اللغة: | English |
| DOI: | 10.1373/clinchem.2004.035808 |
| الاتاحة: | http://dx.doi.org/10.1373/clinchem.2004.035808 http://academic.oup.com/clinchem/article-pdf/50/10/1755/32736882/clinchem1755.pdf |
| Rights: | https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model |
| رقم الانضمام: | edsbas.D295D1CB |
| قاعدة البيانات: | BASE |
| DOI: | 10.1373/clinchem.2004.035808 |
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