One-step site-directed mutagenesis of viroid dimeric cDNA.

One-step site-directed mutagenesis of viroid dimeric cDNA. Research Abstract Details 

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One-step site-directed mutagenesis of viroid dimeric cDNA. Abstract Text:

Rafael , ,

The study of viroids (plant pathogens constituted by small non-coding RNA) has greatly benefited from the use of site-directed mutagenesis tools. However, compared to viral systems, this technique is complicated by the fact that, usually, infectious cDNAs carry two copies of the viroid genome. A simple method for a one-step site-directed mutagenesis of viroids is described and tested by estimating the rate of mutation incorporation of three random nucleotide substitutions in each Chrysanthemum stunt viroid (CSVd) and Chrysanthemum chlorotic mottle viroid (CChMVd). The protocol is essentially based on the original QuickChange Stratagene methodology; dimeric cDNA templates are amplified directly using Pfu DNA polymerase and self-complementary mutagenic primers. The reaction typically yields dimeric, but also monomeric clones which can be easily distinguished by electrophoretic analysis. The data show that approximately 50% of the dimeric clones carry the desired mutation in both viroid copies. Since the proposed protocol is simple technically and rapid compared to previous methods, it could be applied routinely for site-directed mutagenesis studies in viroids.

One-step site-directed mutagenesis of viroid dimeric cDNA. Publishing Authors By Initials

R ,JA ,

For similar viruses: viroids research abstracts see: viruses: viroids research

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One-step site-directed mutagenesis of viroid dimeric cDNA. Journal Published:

PUBLICATION TYPE: Research Support, Non-U.S. Gov

Journal: Journal of virological methods

VOLUME: 145

Page Numbers: 71-5

Journal Abbreviation: J. Virol. Methods

ISSN: 0166-0934

DAY: 29

MONTH: 06

YEAR: 2007

One-step site-directed mutagenesis of viroid dimeric cDNA. Information

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LANGUAGE: eng

NlmUniqueID: 8005839

One-step site-directed mutagenesis of viroid dimeric cDNA. Keywords Mesh Terms:

KEYWORDS: Viroids

MESH TERMS: genetics

Chemical & Substance for Abstract: One-step site-directed mutagenesis of viroid dimeric cDNA. Information

Substance Name: RNA, Viral

Registry Number: 0

Grant and Affiliation Information for One-step site-directed mutagenesis of viroid dimeric cDNA.

AFFILIATION: Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV), C/ Ingeniero Fausto Elio s/n, 46022 València, Spain. rafsaver@ibmcp.upv.es

Country: Netherlands

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MEDLINETA: J Virol Methods

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