Single-tube linear DNA amplification for genome-wide studies using a few thousand cells

Nat Protoc. 2012 Jan 26;7(2):328-38. doi: 10.1038/nprot.2011.447.

Abstract

Linear amplification of DNA (LinDA) by T7 polymerase is a versatile and robust method for generating sufficient amounts of DNA for genome-wide studies with minute amounts of cells. LinDA can be coupled to a great number of global profiling technologies. Indeed, chromatin immunoprecipitation coupled to massive parallel sequencing (ChIP-seq) has been achieved for transcription factors and epigenetic modification of chromatin histones with 1,000 to 5,000 cells. LinDA largely simplifies reChIP-seq experiments to monitor co-binding at chromatin target sites. The single-tube design of LinDA is ideal for handling ultrasmall amounts of DNA (<30 pg) and is compatible with automation. The actual hands-on working time is less than 6 h with one overnight reaction. The present protocol describes all materials and critical steps, and provides examples and controls for LinDA. Applications of LinDA for genome-wide analyses of biobank samples and for the study of chromatin conformation and nuclear architecture are in progress.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Composition
  • Chromatin Immunoprecipitation
  • Genomics / methods*
  • Nucleic Acid Amplification Techniques*
  • Plasmodium falciparum / genetics
  • Sequence Analysis, DNA
  • Transcription, Genetic