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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.
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...
Maxam-Gilbert Sequencing01:05

Maxam-Gilbert Sequencing

In the same year as the discovery of the Sanger sequencing method, another group of scientists, Allan Maxam and Walter Gilbert, demonstrated their chemical-cleavage method for DNA sequencing. The Maxam-Gilbert method relies on using different chemicals that can cleave the DNA sequence at specific sites, the separation of resulting DNA fragments of variable size using electrophoresis, and deciphering the DNA sequence from the resulting gel bands.
Challenges of the Maxam-Gilbert Method
The...

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Updated: Jun 20, 2026

Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing
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Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing

Published on: July 7, 2020

SplinkBES: a splinkerette-based method for generating long end sequences from large insert DNA libraries.

Pablo F Cavagnaro1, Douglas Senalik, Philipp W Simon

  • 1Department of Horticulture, University of Wisconsin, Madison, WI 53706, USA.

Biotechniques
|September 10, 2009
PubMed
Summary
This summary is machine-generated.

A new splinkerette-based sequencing method generates significantly longer bacterial artificial chromosome end sequences (BESs). Longer BESs provide more genomic information, aiding in genomics, mapping, and breeding applications.

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Area of Science:

  • Genomics
  • Molecular Biology
  • Biotechnology

Background:

  • Bacterial artificial chromosome (BAC) libraries are crucial for genome sequencing and analysis.
  • Obtaining long, informative end sequences from BAC clones is essential for efficient genome mapping and characterization.
  • Current methods for BAC end sequencing often yield shorter sequences, limiting their utility.

Purpose of the Study:

  • To develop and validate a novel splinkerette-based method for generating long end sequences from large-insert library clones.
  • To assess the informativeness of longer BAC end sequences (BESs) compared to shorter ones.
  • To propose a high-throughput procedure for this novel sequencing approach.

Main Methods:

  • Developed a splinkerette-based method involving BAC DNA digestion, adaptor ligation, and PCR amplification of BAC ends.
  • Sequenced amplicons from both directions and assembled overlapping sequences into ungapped and gapped BESs.
  • Analyzed BESs from carrot and five other model organisms, comparing actual and in silico-generated sequences of varying lengths.

Main Results:

  • The novel method produced significantly longer BESs (average 838 nucleotides) compared to direct end sequencing.
  • Longer BESs exhibited a higher number of matches in the GenBank database and contained more simple sequence repeats (SSRs).
  • Demonstrated that sequence length directly correlates with the informativeness of BESs for genomic analysis.

Conclusions:

  • The splinkerette-based method effectively generates long and informative BAC end sequences.
  • Longer BESs enhance the accuracy and depth of genomic analysis, including marker development.
  • The proposed splinkerette-based end sequencing (SplinkBES) method offers a promising advancement for high-throughput genomics, mapping, and breeding.