Homopolymeric tailing

Joining DNA by complementary homopolymeric tails Terminal transferase... 

Terminal deoxynucleotidyl transferase (TdT) polymerizes DNA by the same mechanism as DNA polymerases but lacks a requirement for a template DNA strand to direct synthesis (27, 28). TdT prefers single-stranded DNA or duplex DNA containing 3 protruding ends, and is often used in the synthesis of a homopolymeric tail. Nucleotide incorporation is influenced by the divalent cation in the reaction Purine nucleotides are incorporated preferentially in the presence of Mg +, while incorporation of pyrimidine nucleotides is favored in the presence of Co. The use of nucleotide analogs (dideoxynucleotides or cor-dycepin triphosphate) results in the incorporation of a single nucleotide (29). 

A number of suppliers currently market custom-synthesized oligonucleotides containing fluorophores or haptens, and these work well as FISH probes (H. Bass, personal communication), but oligos can also be successfully labeled using the end-labeling scheme described above (Protocol II.B.2). We were initially concerned that the homopolymeric tail added by terminal transferase might interfere with the specificity of the probes (especially because the tail... 

In molecular biology, TDT is used to effect the addition of complementary homopolymeric tails to vector and complementary DNA (cDNA) obtained from a mature mRNA template by reverse transcription and PCR amplification. An alternative application of TDT involves labehng of the 3 termini of DNA fragments with a P-labeled dNTP [36], a dideoxynucleoside triphosphate (ddNTP) [37], or a ribonucleoside triphosphate (rNTP) [38]. 

In an efficient method utilizing the 3 — 5 -exonuclease activity of T4 DNA Pol (30,31), programmed deletions are introduced into the ssDNA which has been linearized by a splinter/adapter-mediated cleavage with a restriction enzyme (Fig. 6.5). The nested sets of fragments are recircularized after homopolymeric tailing via the same splinter/adapter and are used for the transformation of competent . coli cells. 

The third alternative is the homopolymeric tailing of cDNA using terminal deoxynucleotidyltransferase and annealing with the vector carrying complementary homopolymeric tails (see Section IV, this chapter). 

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