T7 Linear Amplification of DNA (TLAD) for Microarrays

Cold Spring Harbor Protocols
Oliver Rando

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.

References

May 13, 2003·BMC Genomics·Chih Long LiuBradley E Bernstein
Aug 27, 2005·PLoS Biology·Chih Long LiuOliver J Rando
Jul 3, 2019·Cold Spring Harbor Protocols·Oliver Rando
Jul 3, 2019·Cold Spring Harbor Protocols·Oliver Rando
Aug 3, 2019·Cold Spring Harbor Protocols·Oliver Rando

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Citations

Jun 7, 2019·Cold Spring Harbor Protocols·Oliver Rando
Aug 3, 2019·Cold Spring Harbor Protocols·Oliver Rando

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