|Publication Type||Journal Article|
|Year of Publication||2008|
|Authors||Liu, CL, Bernstein, BE, Schreiber, SL|
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 T7 phage. TLAD was designed 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. In Part I of the method, described here, a 3' conserved end is added to the template dsDNA, using terminal deoxynucleotidyl transferase (TdT) tailing. The initial treatment with calf intestinal phosphatase (CIP) is optional but strongly recommended for removing 3' phosphate groups, because most genomic DNA fragmentation methods (i.e., sonication, micrococcal nuclease digestion, and certain restriction digests) produce a significant proportion of 3' phosphate groups within the mixture of fragmented genomic DNA. This protocol is compatible with the presence of RNase A and can be carried out immediately after digestion of RNA carried over from ChIP, without any intermediate clean-up step. The tailing reaction involves the addition of a short (20-40 nucleotide [nt]) poly(dT) tail to the template DNA. The included dideoxynucleotide acts as a tail terminator in the reaction mixture and is necessary to maintain a tight size distribution. This poly(dT) tail provides a conserved 3' element that permits the addition of a T7 promoter sequence in the subsequent second-strand synthesis step. IVT can then use this newly appended T7 promoter and linearly amplify the template dsDNA, producing amplified RNA product.