Topic Introduction

Whole Genome Amplification by T7-Based Linear Amplification of DNA (TLAD): Overview

Adapted from "DNA Linear Amplification," Chapter 7, in Whole Genome Amplification: Methods Express (eds. Hughes and Laskin), from the Methods Express series. Scion Publishing Ltd., Oxfordshire, UK, 2005.

INTRODUCTION

T7-based linear amplification of DNA (TLAD) was designed primarily to overcome the shortcomings of exponential amplification approaches. TLAD uses a linear amplification method based on the in vitro transcription (IVT) of template DNA by RNA polymerase from the T7 phage, a common strategy used in RNA amplification protocols. The TLAD protocol introduced here successfully addresses amplification fidelity issues encountered with use of random PCR (R-PCR) with the ChIP-chip method (whereby DNA recovered from chromatin immunoprecipitation [ChIP] of cell lysate is used for subsequent analysis on DNA microarrays). Thus, optimizations have been done with this particular application in mind. For other applications that require DNA instead of RNA to be the end point, reverse transcription is a necessary step that increases the cost and complexity of necessary molecular biological manipulations to the sample, when compared with PCR. However, IVT amplification does offer improved fidelity and a much higher maximum yield per single reaction. Thus, other techniques that require amplification of complex mixtures of randomly fragmented genomic DNA can also benefit from this method.