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Related Concept Videos

RACE - Rapid Amplification of cDNA Ends02:35

RACE - Rapid Amplification of cDNA Ends

Rapid Amplification of cDNA Ends, or RACE, is one of the most effective methods to obtain a full-length cDNA from an mRNA sequence between a known internal region to the unknown sequence at the 5’ or 3’ end. The unknown region is cloned in the cDNA by a gene-specific primer that binds the known end, and a hybrid primer that attaches a predefined anchor sequence to the unknown end of the cDNA. The sequence in between is amplified by PCR with an anchor primer and a gene-specific primer.
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Next-generation Sequencing

The first human genome sequencing project cost $2.7 billion and was declared complete in 2003, after 15 years of international cooperation and collaboration between several research teams and funding agencies. Today, with the advent of next-generation sequencing technologies, the cost and time of sequencing a human genome have dropped over 100 fold.
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Although all next-generation methods use different technologies, they all share a set of standard features.

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A linear concatenation strategy to construct 5'-enriched amplified cDNA libraries using multiple displacement

Vijay J Gadkar1, Martin Filion

  • 1Department of Biology, Université de Moncton, Moncton, Canada.

Molecular Biotechnology
|September 4, 2012
PubMed
Summary

Researchers can now amplify limited RNA samples for large-scale gene analysis using a novel cDNA library construction protocol. This method ensures sufficient material for downstream applications, including 5' RACE analysis.

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Linear Amplification Mediated PCR &#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA
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Published on: June 25, 2014

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Linear Amplification Mediated PCR &#8211; Localization of Genetic Elements and Characterization of Unknown Flanking DNA
11:58

Linear Amplification Mediated PCR – Localization of Genetic Elements and Characterization of Unknown Flanking DNA

Published on: June 25, 2014

Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Limited RNA quantities hinder large-scale gene transcript analysis in experimental systems.
  • Pre-amplification of RNA/cDNA is a strategy to overcome low sample input.
  • Existing methods may not preserve critical RNA end information for all analyses.

Purpose of the Study:

  • To develop a novel protocol for constructing amplified cDNA libraries from limited total RNA.
  • To enable large-scale downstream analyses, including polymerase chain reaction (PCR) and 5' RACE.
  • To ensure the integrity of the 5' ends of gene transcripts in the amplified library.

Main Methods:

  • Utilized the Phi29 DNA polymerase-based multiple displacement amplification (MDA) system.
  • Developed a linear concatenation strategy for single-stranded cDNA template preparation.
  • Employed a template-switching reverse transcriptase for cDNA synthesis.

Main Results:

  • Successfully generated micrograms of amplified cDNA from as little as 200 ng of total RNA.
  • The protocol yielded amplified cDNA libraries with intact 5' ends.
  • Demonstrated protocol efficacy in both characterized (tomato) and uncharacterized (corn gromwell) systems.

Conclusions:

  • The novel MDA-based protocol effectively amplifies limited RNA samples for comprehensive gene analysis.
  • The method provides sufficient cDNA for large-scale PCR and is compatible with 5' RACE.
  • This approach significantly enhances the utility of small RNA samples in transcriptomic studies.