T7 Linear Amplification of DNA (TLAD) for Microarrays

Abstract

Round A/Round B amplification has been used extensively in genomic localization analysis and appears to work quite well for typical microarray applications in which DNA is sheared by sonication to ∼500 bp. For amplification of material <500 bp in size, however, T7 linear amplification of DNA (TLAD) is preferred because it more accurately maintains uniform representation of short DNA fragments. Amplification of double-stranded DNA by TLAD begins with the addition of a 3′ tail of poly-thymidine to DNA by TdT. Second, the Klenow fragment of Escherichia coli DNA polymerase is used, along with a T7-poly(A) primer, to generate a complementary strand that carries a 5′ T7 primer. Finally, extension of the original T-tailed DNA strand yields a template suitable for T7-based transcription, which generates amplified RNA (aRNA). This technique avoids the “jackpotting” issues observed with PCR, in which an early amplification event leads to disproportionate representation of a particular sequence, because PCR follows exponential kinetics, whereas transcription is a linear amplification method.