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Related Experiment Video

Updated: May 31, 2026

Linear Amplification Mediated PCR – 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

Linear amplification for deep sequencing.

Wieteke A M Hoeijmakers1, Richárd Bártfai, Kees-Jan Françoijs

  • 1Department of Molecular Biology, Faculty of Science, Nijmegen Center for Molecular Life Sciences, Radboud University, Nijmegen, The Netherlands.

Nature Protocols
|July 2, 2011
PubMed
Summary

Linear amplification for deep sequencing (LADS) offers a rapid, 2-day method for creating representative sequencing libraries. This technique overcomes PCR bias, ensuring accurate DNA fragment representation for next-generation sequencing, even with limited starting material.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Biotechnology

Background:

  • Standard PCR amplification introduces bias in DNA sequencing libraries, particularly for AT- or GC-rich fragments.
  • Limited starting DNA material poses a challenge for generating sufficient quantities for deep sequencing applications.

Purpose of the Study:

  • To develop a novel amplification method for generating representative sequencing libraries.
  • To overcome the limitations of PCR bias and low DNA input in next-generation sequencing library preparation.

Main Methods:

  • Linear amplification for deep sequencing (LADS) involves attaching distinct adapters to DNA fragments, one with a T7 RNA polymerase promoter.
  • In vitro transcription generates high RNA yields, followed by cDNA synthesis to create full-length libraries.
  • Size selection and adapter ligation are key steps in the LADS protocol.

Main Results:

  • LADS produces representative sequencing libraries within 2 days.
  • T7-amplified libraries exhibit sequence coverage indistinguishable from nonamplified libraries, eliminating PCR bias.
  • LADS successfully generates libraries from nanogram quantities of DNA, suitable for low-input applications.

Conclusions:

  • Linear amplification for deep sequencing (LADS) provides an unbiased and efficient method for next-generation sequencing library preparation.
  • The LADS method is particularly valuable for applications with limited DNA starting material.
  • LADS offers a significant advancement over traditional PCR-based amplification and amplification-free methods.