A Simple and Efficient Method for High Fidelity PCR

Bioscience, Biotechnology, and Biochemistry

ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20

A Simple and Efficient Method for High Fidelity PCR Cloning Using Antibody-neutralizing Technology Masao KITABAYASHI, Yoshiaki NISHIYA & Muneharu ESAKA To cite this article: Masao KITABAYASHI, Yoshiaki NISHIYA & Muneharu ESAKA (2003) A Simple and Efficient Method for High Fidelity PCR Cloning Using Antibody-neutralizing Technology, Bioscience, Biotechnology, and Biochemistry, 67:9, 2034-2037, DOI: 10.1271/bbb.67.2034 To link to this article: https://doi.org/10.1271/bbb.67.2034

Published online: 22 May 2014.

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Biosci. Biotechnol. Biochem., 67 (9), 2034–2037, 2003

Note

A Simple and E‹cient Method for High Fidelity PCR Cloning Using Antibody-neutralizing Technology Masao KITABAYASHI,1,2 Yoshiaki NISHIYA,3 and Muneharu ESAKA1,† 1Graduate

School of Biosphere Sciences, Hiroshima University, Kagamiyama, Higashi-Hiroshima 739-8528,

Japan 2Tsuruga Institute of Biotechnology, Toyobo Co., Ltd., 10-24 Toyo-cho, Tsuruga, Fukui 914-0047, Japan 3Bio-division, Toyobo Co., Ltd., 2-8 Dojima Hama 2-chome, Kita-ku, Osaka 530-8230, Japan Received April 11, 2003; Accepted June 13, 2003

We introduce the TA cloning antibody method for the high-ˆdelity PCR product ampliˆed by family B DNA polymerase without puriˆcation. This method uses antibodies and Thermus aquaticus (Taq ) DNA polymerase. The antibodies can inhibit only the activity of family B DNA polymerase, and Taq can co-work for A-tailing. This method has nearly cloning e‹ciency to that of the PCR product of Taq. Key words:

high-ˆdelity PCR product; family B DNA polymerase; KOD DNA polymerase; TA cloning method; antibody

Advances of biotechnology have enabled the research community to use vast amounts of data at an ever-increasing pace. The comparison of DNA sequences from model organisms, in which genetic manipulations can be easily done, is attempted to clarify gene function. Thus, we require DNA ampliˆcation with PCR to identify the DNA sequence. The family A DNA polymerases including the Thermus aquaticus DNA polymerase (Taq DNA polymerase) are useful for PCR, have no 3? to 5? exonuclease activity, and hence put a lot of mutations into the PCR product. On the other hand, family B DNA polymerases that have a ``proofreading'' function, such as the DNA polymerase from Thermococcus kodakaraensis KOD1 (KOD DNA polymerase), have 3? to 5? exonuclease activity. Therefore, the PCR ˆdelity of KOD DNA polymerase is about 50 times that of Taq DNA polymerase.1,2) However, Taq DNA polymerases are frequently used in PCR cloning because they can be used simply in a convenient cloning system, TA cloning.3) The principle of TA cloning is as follows. The PCR product ampliˆed by a family A DNA polymerase has a deoxyriboadenosine (dA) addition at the 3? end. The linear plasmid, called the T-plasmidvector, has a deoxyribothymine (dT) addition at the 3? end, which is the complementary sequence to the †

dA of the PCR product. Thus, the T-plasmid-vector can be easily ligated with the PCR product. Now we can use the T-plasmid-vectors marketed by a few companies. On the other hand, the PCR product ampliˆed by family B DNA polymerase has a blunt end because of its ``proofreading'' function. The PCR product, therefore, must be ine‹ciently cloned with a vector plasmid by blunt end ligation. We must treat Atailing at the 3? blunt end by Taq DNA polymerase after purifying the ampliˆed DNA fragments as the manufacturer's protocol of pGEM-T (Promega, Madison, WI), if the TA cloning method is used (Fig. 1A). Here, we introduce the TA cloning method for the PCR products ampliˆed by family B DNA polymerases without puriˆcation. This method uses antibodies against a family B DNA polymerase and Taq DNA polymerase at the same time. The antibodies can inhibit only the activity of family B DNA polymerase, and Taq DNA polymerase can work for A-tailing of the PCR product. We named this method the TA cloning antibody method (Fig. 1B). To use the antibodies which can inhibit both activities of DNA polymerase and 3?–5? exonuclease having family B DNA polymerase was required to succeed with the TA cloning antibody method. We experimented to prove this method as follows. PCR was done in 50 ml by lambda DNA (1 ng) as a template, sense and anti-sense primers (15 pmol each), and the Taq or KOD DNA polymerase according to each manufacturer's instructions. Two primers designed were as follows: lambda-sense (5?-GATGAGTTCGTGTCCGTACAACT-3?) and lambdaanti-sense (5?-GATAGCTGTCGTCATAGGACTC3?). Only a 2-kb DNA fragment was obtained without extra ampliˆcations. We did TA cloning of the PCR product. We tried to experiment with the TA cloning antibody method using antibodies recognizing KOD DNA polymerase (anti-KOD antibodies)

To whom correspondence should be addressed. Fax: ＋81-824-24-7927; E-mail: mesaka＠hiroshima-u.ac.jp

TA Cloning Antibody Method for High Fidelity PCR

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Fig. 1.

Comparison of the TA Cloning Method. A) The puriˆcation and the A-tailing reaction are usually required for TA cloning of the PCR product ampliˆed by a family B DNA polymerase. B) The TA cloning antibody method requires no puriˆcation of the PCR product ampliˆed by a family B DNA polymerase. C) The PCR product ampliˆed by Taq DNA polymerase is directly reacted with T-plasmid-vector.

and Taq DNA polymerase.4) The stable temperature C) of anti-KOD antibodies was greatly diŠerent (379 from the optimal temperature (729 C) of Taq DNA polymerase. We investigated the temperature by which anti-KOD antibodies and Taq DNA polymerase can work at the same time. Three hundred and thirty ng of anti-KOD antibodies and 5 units of the Taq DNA polymerase were directly added to the reaction mixture after PCR, and reacted for 30 minutes at various temperatures. A portion (3 ml) of the reaction mixtures was used to ligate into a T-plasmid-vector, pGEM-T, at 259C for 60 min according to the manufacturer's protocol. Ten ml of the ligation mixture was used to transform Escherichia coli DH5a competent cells (Toyobo).5) One hundred ml of transformed cells were spread onto LB plates containing 5-bromo-4-chloro-3-indolyl-b-D-galactoside and ampicillin, and then were cultured at 379 C overnight. The cloning e‹ciency was calculated as the ratio of white colonies to all blue W white colonies. As a result, the best reaction temperature of this method was 609 C (Fig. 2A). The cloning e‹ciency was not enough (Method 3, 6.2z, Fig. 2B), when only 5 U of Taq DNA polymerase was added to the PCR product ampliˆed with KOD DNA polymerase and it was processed at 609 C. On the other hand, the cloning e‹ciency was about 45z (Method 4, Fig. 2B), when the DNA fragment A-tailed by using the anti-KOD antibodies and the Taq DNA polymerase were used for the TA cloning. And the

cloning e‹ciency was about 41z (Method 5, Fig. 2B), when the DNA fragment A-tailed by the 5 units of Taq DNA polymerase for 30 min at 709C in 10 ml of 1×buŠer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 0.1z TritonX-100, 1.5 mM MgCl2 and 0.2 mM dATP) after puriˆcation of the PCR product, was used for the TA cloning. These two methods (Method 4 and 5) were almost the same cloning e‹ciency and about ten times that of untreated method (Method 2, 4.6z, Fig. 2B). Further this method (Method 4) without puriˆcation has nearly the same cloning e‹ciency as that (Method 1, 52z, Fig. 2B) of the PCR product of Taq DNA polymerase, and there was no problem in practical use. We did colony-direct PCR with KOD-Plus DNA polymerase using transformants obtained by Method 4 to conˆrm the existences of insert DNA (Fig. 2C).6) The insert DNA was found in all of ˆve white colonies randomly selected. When the blunt-end cloning method was done using the Perfectly Blunt Cloning Kits (Novagen, Madison, WI), its cloning e‹ciency (43z) was almost the same as that of the TA cloning antibody method. However, the number of white colonies obtained in the blunt end cloning method 10 (245 colonies), while white colonies was about 1 W (2320 colonies) in Method 4 were not very diŠerent from white colonies (2360 colonies) in Method 1. Moreover the clones connected mutually several PCR fragments that appeared occasionally, when short PCR products were cloned with this method.

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Fig. 2.

Cloning E‹ciency of TA Cloning Antibody Method. white colony selection. (A) The optimal temperature of TA cloning antibody method. The cloning e‹ciency was evaluated by blue W The PCR product ampliˆed by KOD DNA polymerase was treated with anti-KOD antibodies and Taq DNA polymerase for 30 min. (B) Comparison of the cloning e‹ciency in four TA cloning methods. Method 1, TA cloning of PCR product with Taq DNA polymerase; Method 2, TA cloning of PCR product with KOD DNA polymerase; Method 3, TA cloning of the DNA fragment treated with A-tailing by Taq DNA polymerase after purifying the PCR product with KOD DNA polymerase; Method 4, TA cloning of the PCR product with KOD DNA polymerase treated with A-tailing by anti-KOD antibodies and Taq DNA polymerase. (C) Result of colony direct PCR using transformants obtained by Method 4. Lane M; size markers (l DNA digested with Hin d III), Lane B; ampliˆcation from a blue colony, Lanes 1¿5; ampliˆcation from white colonies.

The TA cloning antibody method required only one manual operation, in which the antibodies and Taq DNA polymerase are added to the reaction mixture after PCR. So this method is not too complicated as compared with the standard TA cloning method (Fig. 1C). The rate of mutation introduction did not increase in the vicinity of either end especially, although we have already used the TA cloning antibody method for PCR products having various sizes (¿9 kb) and sequences (data not shown). We tried the TA cloning antibody method for the same 2-kb DNA fragment ampliˆed with the Pfu DNA polymerase, which belonged to the family B DNA polymerases. The monoclonal antibodies against Pfu DNA polymerase contained in Pfutubo Hotstart DNA polymerase (Stratagene, La Jolla, CA) were used in this experiment. It was possible to obtain a su‹cient cloning e‹ciency (35z), when the antibodies against Pfu DNA polymerase and the Taq DNA polymerase were used. And we compared it to the method with an A-addition kit (Qiagen, Darmstadt, Germany) which did A-tailing at 379C for the PCR product ampliˆed with all family B DNA polymerase. The cloning e‹ciency was not enough (9.0z), when the same 2-kb DNA fragment ampliˆed with KOD DNA polymerase was processed with the A-addition kit. The principle of this kit has not been clariˆed. We thought that our method had two merits. One was that each of the antibodies against family B DNA polymerase could surely inhibit the activities of each family B DNA polymerase. And another was that Taq DNA polymerase could add dA in high e‹ciency selectively. So, the TA

cloning antibody method could get a higher cloning e‹ciency than that of the A-addition system, and it worked as well as that of the PCR product ampliˆed with Taq DNA polymerase. In summary, we developed the simple and e‹cient method named the TA cloning antibody method, for cloning of a high-ˆdelity PCR product ampliˆed with family B DNA polymerase. The TA cloning antibody method exploited the neutralizing technology with the antibodies, and required no DNA puriˆcation. We believe that the TA cloning antibody method can cancel the faults in the cloning method of family B DNA polymerases.

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