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

Nature protocols (2012-01-28)
Pattabhiraman Shankaranarayanan, Marco-Antonio Mendoza-Parra, Wouter van Gool, Luisa M Trindade, Hinrich Gronemeyer
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.

MATERIALS
Product Number
Brand
Product Description

Roche
Taq DNA Polymerase, 5 U/μl, optimum pH ~9.0 (20 °C), optimum reaction temp. 72 °C