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Related Concept Videos

Next-generation Sequencing03:00

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.
Next-Generation Sequencing Methods
Although all next-generation methods use different technologies, they all share a set of standard features.
RNA-seq03:21

RNA-seq

RNA sequencing, or RNA-Seq, is a high-throughput sequencing technology used to study the transcriptome of a cell. Transcriptomics helps to interpret the functional elements of a genome and identify the molecular constituents of an organism. Additionally, it also helps in understanding the development of an organism and the occurrence of diseases. 
Before the discovery of RNA-seq, microarray-based methods and Sanger sequencing were used for transcriptome analysis. However, while microarray-based...
Sanger Sequencing01:57

Sanger Sequencing

DNA sequencing is a fundamental technique that is routinely used in the biological sciences. This method can be applied to a range of questions at different scales - from the sequencing of a cloned DNA fragment or the study of a mutation in a gene up to whole-genome sequencing. However, despite the widespread use of sequencing today, it was not until 1977 that Fredrick Sanger and his collaborators developed the chain-termination method to decode DNA sequences. It relies on the separation of a...
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.
Since the...

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Related Experiment Video

Updated: Jul 3, 2026

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach
11:12

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Published on: September 11, 2017

A high-throughput genome-walking method and its use for cloning unknown flanking sequences.

Palakolanu Sudhakar Reddy1, Srikrishna Mahanty, Tanushri Kaul

  • 1International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110 067, India.

Analytical Biochemistry
|August 5, 2008
PubMed
Summary

This study introduces a novel, high-throughput genome walking method using PCR and Phi29 DNA polymerase. The technique efficiently maps unknown DNA regions, enabling the isolation and sequencing of gene flanking sequences.

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Novel Sequence Discovery by Subtractive Genomics
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Novel Sequence Discovery by Subtractive Genomics

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Last Updated: Jul 3, 2026

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach
11:12

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Published on: September 11, 2017

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome
06:40

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

Published on: March 22, 2018

Novel Sequence Discovery by Subtractive Genomics
09:40

Novel Sequence Discovery by Subtractive Genomics

Published on: January 25, 2019

Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Genome walking is crucial for identifying regulatory elements and gene structures.
  • Existing methods can be time-consuming and lack efficiency for high-throughput applications.

Purpose of the Study:

  • To develop a novel, efficient, and high-throughput PCR-based genome walking protocol.
  • To enable the rapid isolation and sequencing of flanking DNA regions, such as promoters.

Main Methods:

  • Utilized rolling circle DNA synthesis with Phi29 DNA polymerase to randomly introduce walker primer binding sites.
  • Employed degenerate primers and strand-displacement activity for comprehensive genomic fragment amplification.
  • Performed directional genome walking using locus-specific and walker primers in successive PCR rounds.

Main Results:

  • Successfully generated overlapping genomic fragments with unique walker adapters.
  • Demonstrated directional genome walking capabilities using locus-specific primers.
  • Isolated and sequenced 5' flanking regions and promoters of selected plant genes with high efficiency.

Conclusions:

  • The developed protocol offers a robust and efficient solution for high-throughput genome walking.
  • This method significantly advances the ability to characterize gene regulatory elements in various organisms.
  • Facilitates rapid gene discovery and functional genomics research.