Whole Genome Amplification by T7-Based Linear Amplification of DNA (TLAD): II. Second-Strand Synthesis and In Vitro Transcription.

CSH protocols

INTRODUCTIONT7-based linear amplification of DNA (TLAD) uses a linear amplification approach based on in vitro transcription (IVT) of template DNA by RNA polymerase from the T7 phage. TLAD was designed primarily for use with the ChIP-chip method (whereby DNA recovered from chromatin immunoprecipitation [ChIP] of cell lysate is used for subsequent analysis on DNA microarrays) and requires nanogram quantities of dsDNA to generate microgram amounts of amplified RNA. Briefly, the strategy is to add a 3′ conserved end to the template dsDNA, using terminal deoxynucleotidyl transferase (TdT) tailing, which permits the addition of a T7 promoter sequence in the subsequent second-strand synthesis step, described here. At this stage, the strand-displacement activity of the Klenow fragment polymerase separates the two strands of the template DNA, after which the enzyme performs fill-in 5′ → 3′ polymerization. Its 3′ → 5′ exonuclease activity may also remove the 3′ overhanging poly(dT) tails, although the efficiency of this activity will vary based on the length of the poly(dT) tail. IVT can then use this newly appended T7 promoter. Because the T7-based IVT proceeds as an isothermal reaction, it linearly amplifies the template DNA, producing antisense RNA (aRNA) (i.e., each strand of RNA produced is antisense to the original template strand). Since both strands are amplified, this distinction is usually not important and is affected only by the location of the T7 promoter and poly(A) tract on the aRNA.