易错PCR

Creating Random Mutagenesis Libraries

by Megaprimer PCR of Whole Plasmid (MEGAWHOP)

Kentaro Miyazaki

1. Introduction

The conventional method for cloning a DNA fragment is to insert it into a vector and ligate it (1). Although this method is commonly used, it is labor intensive because the ratio and concentrations of the DNA insert and the vector must be optimized. Even then, the resulting library is often plagued with unwanted plasmids that have no inserts or multiple inserts. These species have to be eradicated to avoid tedious screening, especially when producing random mutagenesis libraries.

MEGAWHOP is a novel cloning method of DNA fragments which was developed as a substitute for the problematic ligation approach (2). In MEGAWHOP, the DNA fragment to be cloned is used as a megaprimer that replaces a homologous region in the template plasmid (see Fig. 1). After run-ning whole plasmid PCR using the megaprimer, the resultant mixture is treated with a dam-methylated DNA specific restriction enzyme, Dpn I. The treatment enables specific elimination of the template plasmid, because the template plas-mid, which can be propagated in most Escherichia coli strains, is dam-methy-lated. The Dpn I-treated mixture is then introduced into E. coli to yield a library. Libraries produced by the MEGAWHOP method are virtually free from con-tamination by species without any inserts or with multiple inserts. A MEGAWHOP protocol that is especially ideal for creating random mutagen-esis libraries is described below. The protocol guarantees a good outcome with a 500–1000 bp megaprimer and a 3–7 kbp template plasmid.

From: Methods in Molecular Biology, vol. 231: Directed Evolution Library Creation: Methods and Protocols Edited by: F. H. Arnold and G. Georgiou ? Humana Press Inc., Totowa, NJ

2. Materials

https://www.360docs.net/doc/6613951289.html,petent cells of E. coli (dam+ strain, e.g., JM109, XL10).

2.Growth medium for E. coli (e.g., Luria Broth (LB), 2xYT).

3.Appropriate antibiotics (e.g., ampicillin, kanamycin, chloramphenicol, tetracycline).

4.DNA plasmid template of known concentration propagated in E. coli dam+strain

and prepared using standard protocols (1).

5.Dpn I restriction enzyme (New England Biolabs; Beverly, MA).

6.10X PfuTurbo?DNA polymerase buffer: 200 m M Tris-HCl, pH 8.8, 100 m M

KCl, 100 m M(NH4)2SO4, 20 m M MgSO4, 1% Triton?X-100, 1 mg/mL bovine serum albumin (Stratagene; La Jolla, CA).

7.PfuTurbo? DNA polymerase (Stratagene).

8.dNTP set (Amersham; Piscataway, NJ).

9.Reagents and apparatus for agarose gel electrophoresis.

10.Qiaquick PCR purification kit (Qiagen, Hilden, Germany).

11.Thermal cycler (e.g., GeneAmp? 9700, Applied Biosystems; Foster City, CA).

3. Methods

The MEGAWHOP cloning method consists of four steps: (1) preparation of the DNA fragment to be cloned (megaprimer); (2) whole plasmid PCR using the megaprimer; (3) Dpn I-treatment of the whole plasmid PCR product; (4) and transformation, as described under Subheadings 3.1–3.4.

3.1. Preparation of DNA Fragment to be Cloned (Megaprimer)

1.Prepare a mutated gene fragment by random mutagenesis, using either a PCR-

based procedure, e.g., error-prone PCR (3), DNA shuffling (4), or StEP recombi-nation(5) (see Note 1) or a non-PCR-based procedure, e.g., chemical mutagenesis

(1). Dissolve the DNA in water (or in Tris-EDTA (TE) buffer).

2.Determine the concentration of the DNA either by spectrophotometry (1 OD260 =

50μg/mL) or by analytical agarose gel electrophoresis (use known concentration of DNA as standard).

3.2. Whole Plasmid PCR

1.Mix megaprimer and plasmid template: 0.5 μg megaprimer (see Note 2), 50 ng

template plasmid (see Note 3), 0.2 m M of each dNTP, and 2.5 U of PfuTurbo?Fig. 1. (opposite)Outline of the MEGAWHOP method. First, the mutated gene fragment is prepared by random mutagenesis. This can be done either by PCR-based (e.g., error-prone PCR, DNA shuffling, StEP recombination) or non-PCR-based pro-cedures (e.g., chemical mutagenesis) (A)Fragments are then annealed to a template plasmid propagated in E. coli dam+strain(B)Next, whole plasmid PCR amplification is carried out to synthesize a nicked circular plasmid (C)The product is then treated with Dpn I to eliminate the template plasmid (D)The Dpn I-treated mixture is then introduced into E. coli and nicks are repaired in vivo (E).

DNA polymerase (see Note 4) in 1x PfuTurbo ?DNA polymerase buffer in a total volume of 50 μL.

2.Run whole plasmid PCR (see Note 5). Incubate the reaction mixture at 68°C for 5 min (this step is optional, see Note 1) and heat at 95°C for 1 min. Perform 18cycles of incubation (see Note 6) at 95°C for 30 s, 55°C for 30 s, and 68°C for 6min, using a GeneAmp ?9700 thermal cycler or equivalent apparatus. You can monitor the reaction by agarose gel electrophoresis as shown in Fig. 2.

3.3.Dpn I Treatment

1.Following whole plasmid PCR, place the reaction on ice for 5 min to cool the reaction below 37°C.

2.Add 20 units of Dpn I (1 μL) directly into the whole plasmid PCR mixture (see Notes 7and 8). Gently and thoroughly mix the reaction mixture by pipetting the solution up and down several times.

3.Incubate for 1–2 hr at 37°C.

3.4. Transformation

https://www.360docs.net/doc/6613951289.html,e 1–2 μL of the Dpn I-treated mixture to transform 20–100 μL of competent E. coli cells (see Note 9) using standard protocols (1). Grow the cells on an agar plate containing appropriate antibiotics.

2.Typically, the transform should yield ~500–1000 colonies when “standard” (i.e.,107/μg of pUC18) competent cells are used. Each plasmid (> 99.9%) should now be carrying a single insert.

4. Notes

1.If you prepare a fragment using Taq DNA polymerase (common in error-prone PCR), be sure to remove extra nucleotides at the 3'-end of the products (7,8)to avoid unintended mutation during whole plasmid PCR. This can be done by treat-Fig.

2. Agarose gel electrophoresis of the MEGAWHOP reaction. 3 μL of the 50 μL

whole plasmid PCR (lane 1, before; lane 2, after) were loaded onto a 1% agarose gel.

ing the fragment with a DNA polymerase having 3' to 5' exonuclease activity

(e.g., T4 DNA polymerase, Pfu DNA polymerase). More conveniently, you can

simply add a short incubation period (68°C for 5 min) prior to the whole plasmid PCR program because the reaction is usually performed with a high-fidelity poly-merase having 3' to 5' exonuclease activity.

2.Increasing the concentration of megaprimer increases the yield of synthesized

plasmid and the number of transformants. The yield reaches a plateau when 0.2 μg of megaprimer (~ 750 bp) and 50 ng of plasmid template (~ 3.5 kbp) are used. No significant increase in the yield is observed even if a larger amount of megaprimer is used.

3.Increasing the concentration of template plasmid increases the yield of synthe-

sized plasmid and the number of transformants. The yield reaches a plateau when

25 ng of template plasmid (~ 3.5 kbp) and 0.5 μg of megaprimer (~ 750 bp) are

used. No significant increase in the yield is observed even if a larger amount of template plasmid is used.

4.In order to minimize the incorporation of new mutations during the long-range

PCR, it is essential to use a high fidelity DNA polymerase. We used PfuTurbo?DNA polymerase. You may also use other types of high fidelity DNA polymerase such as native Pfu DNA polymerase (Stratagene), Vent?DNA polymerase (New England Biolabs), or KOD DNA polymerase (Toyobo; Tokyo, Japan).

5.The extension time can be calculated as 2 min/kb, based on the length of the

DNA template using PfuTurbo?DNA polymerase. If you use a different DNA polymerase, the time has to be optimized. Follow the instructions supplied with the polymerase you choose.

6.Increasing the number of cycles increases the yield of synthesized plasmid and

the number of transformants. In our case, decreasing the number of cycles to 12 decreases the yield of transformants to approximately one-third. On the other hand, increasing the number of cycles to 24 increases the yield of transformants about twofold.

7.DpnI digests only methylated and hemi-methylated DNA (6). Therefore, newly

synthesized DNA using “standard” dNTP will not be digested by the Dpn I. On the other hand, template plasmid propagated in common E. coli is dam-methy-lated and hence will be digested with the restriction enzyme.

8.Most PCR buffers do not interfere with the Dpn I reaction.

9.The maximum yield of transformants is obtained when 0.5 μg of megaprimer

(~750 bp) and 50 ng of template plasmid (~ 3.5 kbp) are used in 24 cycles of whole plasmid PCR. Under these conditions, more than 1 ×105transformants are routinely obtained by using 1 μg of megaprimer and 100 μL of standard E. coli competent cells (~107/μg of pUC18). Virtually all (>99.9%) of the screened clones should contain a single insert.

Acknowledgments

I thank Misa Takenouchi for technical assistance. I also thank Professor Frances Arnold and her colleagues for helpful discussions.

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