42. A New Technique to Prevent Self-Ligation of DNA

Hideki Ukai¹, Maki Ukai-Tadenuma², Toshiaki Ogiu and Hideo Tsuji
(¹Domestic Postdoctoral Fellow; ²Japan Science and Technology Corporation)

Keywords: gene cloning, self-ligation, molecular technique

Ligation of DNA fragments by DNA ligase is an essential step in many molecular biology techniques, including gene cloning and messenger RNA (mRNA) fingerprinting. Efficient ligation requires the DNA strands to be prevented from self-ligating (self-circularization and concatenation). Here, we describe a technique to prevent self-ligation of the DNA ligation-partner of 5'-dephosphorylated DNA.

Some techniques to prevent self-ligation are already known, including standard dephosphorylation of the 5' ends by alkaline phosphatase. Because DNA ligase catalyzes the formation of the phosphodiester bond between the 5'-phosphate group (5'-P) and the 3'-hydroxyl group (3'-OH), 5'-dephosphorylated DNA can not self-ligate. The 5'-dephosphorylated DNA still contains a 3'-OH, however, allowing ligation with untreated DNA that contains a 5'-phosphate group (5'-P). The untreated DNA ligation-partner remains capable of self-ligation, e.g., concatenated DNA fragments could insert into the 5'-dephosphorylated vector DNA molecule.

Another technique used to prevent self-ligation is partial filling-in. In this technique, the two DNA species are digested with different restriction enzymes that form 3'-recessed ends. The ends are then partially filled-in using Klenow DNA polymerase with deoxy-nucleotides (dNTPs). The resulting ends are unable to self-ligate but ligate with partner DNA via the complementary 5'-overhang sequence. Although this technique simultaneously prevents self-ligation of both DNA species, it cannot be applied to 5'-recessed or blunt ends. Moreover, only restriction enzymes that produce complementary overhang sequences after partial filling-in can be used, further reducing the flexibility of this technique.

The ligation reaction catalyzed by DNA-ligase is similar to the polymerization of nucleotides by DNA polymerase. DNA polymerase catalyzes the formation of the phosphodiester bond between the 5'-P of a mononucleotide and the 3'-OH of a polynucleotide. A standard technique to inhibit the extension of DNA by DNA polymerase employs dideoxyribonucleotides (ddNTPs). When DNA has a ddNTP incorporated at its 3'-end, the lack of a 3'-OH prevents DNA polymerase from extending the DNA.

In the present study, we replaced the 3'-dNTP of DNA with ddNTP to prevent self-ligation by DNA ligase (Fig. 23). The resulting 3'-H DNA fragment, denoted here as a 3'-replaced DNA fragment, was unable to self-ligate in the presence of DNA ligase. Moreover, because the 3'-replaced fragment still contained a 5'-P, the fragment remained capable of ligating with the 3'-OH of the partner DNA. By combining this 3'-replacement technique with the 5'-dephosphorylation technique, self-ligation of both species of DNA involved in a given ligation was simultaneously prevented, while the 5'-phosphate remaining at the 5'-end permitted ligation with the 3'-hydroxyl end of the 5'-dephosphorylated DNA strand. We successfully applied this 3'-replacement technique to gene cloning, adapter-mediated polymerase chain reaction, and messenger RNA fingerprinting. The 3'-replacement technique is simple and not restricted by sequence or conformation of the DNA termini, and is thus applicable to a wide variety of methods involving ligation.

Fig.23. Prevention of DNA ligation by 3'-replacement. (A) Patterns of DNA ligation. DNA ligase catalyzes the phosphodiester bond (dotted lines) between a 5'-phosphate group (5'-P) and a 3'-hydroxyl group (3'-OH). (a) When DNA contains both the 5'-phosphate and 3'-hydroxyl groups, both strands of DNA are ligated. DNA that does not contain a (b) 5'-phosphate group or a (c) 3'-hydroxyl group cannot self-ligate. (d) 3'-replaced DNA contained a 5'-P, which would be able to ligate with the 3'-OH of the partner DNA, even though the partner was 5'-dephosphorylated. (B) Scheme of the 3'-replacement method. Restriction enzyme-digested DNA was blunt-ended by T4 DNA polymerase and a ddNTP mix that contained one type of ddNTP and three types of dNTPs. The ddNTP was the complementary nucleoside to the 5'-end of the restriction enzyme-digested strand. Immediately after incorporation of the ddNTP, it was removed via the 3'-5' exonuclease activity of T4 DNA polymerase. Thus, incorporation and removal of the ddNTP were repeated at the DNA terminus. The DNA was then incubated with Taq DNA polymerase, which lacks 3'-5' exonuclease activity, at 72 C. Under these conditions, T4 DNA polymerase was completely inactivated, and the Taq DNA polymerase filled the recessed 3' terminus with ddNTP without a 3'-dAMP overhang.