Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynudeotidyl transferase

Abstract

Terminal deoxynucleotidyl transferase, which requires a single-stranded DNA primer under the usual assay conditions, can be made to accept double-stranded DNA as primer for the addition of either rNMP or dNMP, if Mg+2 ion is replaced by Co+2 ion. The priming efficiency in the presence of (C leads to) CO+2 ion with respect to initial rate tested with 2 single-stranded primer, is 5-6 fols higher than that observed with Mg+2 ion. In the presence of Co+2 ion, the primer specificity is altered so that all forms of duplex DNA molecules can be labeled at their unique 3' -ends regardless of whether such ends are staggered or even. Thus, using ribonucleotide incorporation, we have for the first time employed this reaction for sequence analysis of duplex DNA fragments generated by restriction endonuclease cleavages. Furthermore, by using Co+2 ion, it is possible to add a long homopolymer tract of deoxyribonucleotides to the 3'-terminus of double-stranded DNA. Therefore, without prior treatment with lambda exonuclease to expose the 3' terminus as single-stranded primer, this reaction now permits insertion of homopolymer tails at the 3'-ends of all types of DNA molecules for the purpose of in vitro construction of recombinant DNA.

Genetic recombination and complementation between bacteriophage T7 and cloned fragments of T7 DNA.

“…At least 11 representatives of a clone that covers the right end of gene 6 were also identified in this set of clones. However, three other types, covering parts of genes 4 (Fig.…”

“…Plasmid pmB9 (7) was used as the cloning vehicle. Fragments of T7 DNA were inserted at the single EcoRI cleavage site of pBM9 using poly(dA-dT) connectors (8)(9)(10)(11). Fragments of T7 DNA were generated either by cleavage with Hpa I (12) or by shearing the DNA to an average size of 4% its full length.…”

Substrate Properties of Fluorescent Ribonucleotides in the Terminal Transferase-Catalyzed Labeling of DNA Sequencing Primers

“…Within the deoxy-series, a base-dependent variation in the tailing efficiency (as defined by the length of the product) was observed (2). For unmodified rNTPs, the use of Co ++ as an alternative cation to Mg ++ stimulated both the yield of rNTP addition (11) as well as the number of residues added. However, with modified rNTPs, the general conclusion was drawn that "only a few (usually one) nucleotides are incorporated" (6).…”

“…Whereas primers T3, F and R had incorporation yields not differing greatly from those observed with the (T) 16 N primers, oligonucleotide SP6 was labeled less efficiently ( Table 4). The secondary structure of DNA is known to have an effect on the addition efficiency of dNTPs (11). However, a computer prediction of potential hairpin structures in these four primers does not lead to a simple correlation to the labeling efficiency (Table 4).…”

“…Following the analyses with sets of ribonucleotides bearing the same fluorescent tag, it has become clear that, as in the case of biotin-dNTPs (2), the nature of the base has some effect on the efficiency and extent of the reaction, and for none of the nucleotides was the incorporation comparable to unmodified rNTPs (11). However, the label attached to the base is a critical determinant in affecting the substrate properties of the nucleotide; the Texas Red label inhibited incorporation, while fluorescein was able to stimulate multiple additions.…”