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Next-generation Sequencing03:00

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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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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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Real Time RT-PCR

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Related Experiment Video

Updated: May 9, 2026

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells
13:07

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Published on: January 30, 2019

Detection of reverse transcriptase termination sites using cDNA ligation and massive parallel sequencing.

Lukasz J Kielpinski1, Mette Boyd, Albin Sandelin

  • 1Department of Biology, University of Copenhagen, Copenhagen, Denmark.

Methods in Molecular Biology (Clifton, N.J.)
|July 23, 2013
PubMed
Summary
This summary is machine-generated.

This study introduces a new method for detecting reverse transcriptase termination sites using CircLigase and Illumina sequencing. This approach enhances throughput for RNA analysis and gene expression studies.

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Area of Science:

  • Molecular Biology
  • Genomics
  • RNA Biology

Background:

  • Reverse transcriptase termination site detection is crucial for RNA structural probing, 5' rapid amplification of cDNA ends (5' RACE), cap analysis of gene expression, and identifying RNA modifications and protein-RNA cross-links.
  • Massive parallel sequencing technologies offer increased throughput for these applications.

Purpose of the Study:

  • To develop a versatile method for detecting reverse transcriptase termination sites using high-throughput sequencing.
  • To enable efficient and accessible analysis of sequencing data for researchers without extensive bioinformatics expertise.

Main Methods:

  • A novel method involving the ligation of an adapter to the 3' end of cDNA using bacteriophage TS2126 RNA ligase (CircLigase™).
  • Subsequent PCR amplification to introduce Illumina adapters and index sequences for pooled sequencing on standard Illumina platforms.
  • Demonstration of read mapping and data analysis using freely available bioinformatics tools.

Main Results:

  • Successful application of the method for detecting transcription start sites in mouse liver cells.
  • The method allows for high-throughput detection of reverse transcriptase termination sites.
  • Accessible data analysis pipelines are provided.

Conclusions:

  • The described method provides a versatile and high-throughput approach for detecting reverse transcriptase termination sites.
  • This technique facilitates various RNA analysis applications, including gene expression studies.
  • The provided analysis tools make the method accessible to a broader research community.