دورية أكاديمية
أعرض في EDS

Optimization of overlap extension PCR for efficient transgene construction.

التفاصيل البيبلوغرافية
العنوان: Optimization of overlap extension PCR for efficient transgene construction.
المؤلفون: Hilgarth RS; Vector Core, Biomedical Research Core Facilities, University of Michigan, Ann Arbor, MI, 48109, United States., Lanigan TM; Division of Rheumatology, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, 48109, United States.; Vector Core, Biomedical Research Core Facilities, University of Michigan, Ann Arbor, MI, 48109, United States.
المصدر: MethodsX [MethodsX] 2019 Dec 04; Vol. 7, pp. 100759. Date of Electronic Publication: 2019 Dec 04 (Print Publication: 2020).
نوع المنشور: Journal Article
اللغة: English
بيانات الدورية: Publisher: Elsevier B.V Country of Publication: Netherlands NLM ID: 101639829 Publication Model: eCollection Cited Medium: Print ISSN: 2215-0161 (Print) Linking ISSN: 22150161 NLM ISO Abbreviation: MethodsX Subsets: PubMed not MEDLINE
أسماء مطبوعة: Original Publication: Amsterdam : Elsevier B.V., [2014]-
مستخلص: PCR is a powerful tool for generating specific fragments of DNA that can be used to create gene variations or tagged expression constructs. Overlap extension PCR is a valuable technique that is commonly used for cloning large complex fragments, making edits to cloned genes or fusing two gene elements together. After difficulties in utilizing this technique following existing methods, we developed an optimized protocol. To accomplish this, three significant changes were made; 1) touchdown PCR cycling parameters were used to eliminate the need for optimizing PCR cycling conditions, 2) the high-fidelity, high-processivity Q5 DNA polymerase was used to improve full-length amplification quality, and 3) a reduced amount of primer in the final PCR amplification step decreased non-specific amplimers. This modified protocol results in consistent generation of gene fusion products, with little to no background and enhanced efficiency of the transgene construction process.
Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(© 2019 The Authors.)
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فهرسة مساهمة: Keywords: Overlap extension PCR; Restriction enzyme free gene splicing; Subcloning
تواريخ الأحداث: Date Created: 20200206 Latest Revision: 20231113
رمز التحديث: 20231215
مُعرف محوري في PubMed: PMC6992990
DOI: 10.1016/j.mex.2019.12.001
PMID: 32021819
قاعدة البيانات: MEDLINE
الوصف
تدمد:2215-0161
DOI:10.1016/j.mex.2019.12.001