A new technique to prevent self-ligation of DNA
Introduction
Ligation of DNA fragments by DNA ligase is an essential step in many molecular biologic techniques, including gene cloning and messenger RNA (mRNA) fingerprinting (Ivanova and Belyavsky, 1995, Kato, 1995, Kato, 1996, Habu et al., 1997, Kohroki et al., 1999, Ivashuta et al., 1999). Efficient ligation requires the DNA strands 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 have already existed, including standard dephosphorylation of the 5′ ends by alkaline phosphatase (Ullrich et al., 1977, Seeburg et al., 1977). Because DNA ligase catalyzes the formation of the phosphodiester bond between the 5′-phosphate group (5′-P) and the 3′-hydroxyl group (3′-OH) (Weiss et al., 1968), 5′-dephosphorylated DNA cannot 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 (Zabarovsky and Allikmets, 1986). 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, thus 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 (So and Downey, 1970). A standard technique to inhibit the extension of DNA by DNA polymerase employs dideoxyribonucleotides (ddNTPs) (Sanger et al., 1977). 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. 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. 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, including gene cloning, adapter-mediated polymerase chain reaction (PCR) (Riley et al., 1990, Rosenthal and Jones, 1990) and mRNA fingerprinting.
Section snippets
Nucleotides
Deoxy-nucleotides (dNTPs), dideoxyribonucleotides (ddNTPs) and oligonucleotides were purchased from Invitrogen (Carlsbad, CA), Takara-Shuzo Co. Ltd. (Kyoto, Japan) and Hokkaido System Science Co. (Hokkaido, Japan), respectively. 5′-Dephosphorylated vector DNA (pBR322, pUC118/HincII-digested) was purchased from Takara Shuzo. The 4.5-kb DNA fragment derived from the mCort gene was a kind gift from Dr Katsube, National Institute Radiological Sciences of Japan. Total RNA samples for mRNA
Principle for prevention of DNA self-ligation by 3′-replacement
DNA ligase catalyzes the formation of the phosphodiester bond between a 5′-P and a 3′-OH. Thus, DNA in which the 3′-OH is replaced with a 3′-H would be unable to self-ligate (Fig. 1A). Moreover, because the 3′-replaced fragment still contained 5′-P, the fragment remained capable of ligating with the 3′-OH of the partner DNA, even though the partner was 5′-dephosphorylated. To prevent self-ligation, we propose a simple technique in which the deoxy-ribonucleotide (dNTP) at the 3′-end is replaced
Acknowledgements
We are indebted to Dr Takanori Katsube for providing the plasmid pmCort encoding mCort gene.
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