David Wilson and Tony Keefe, March 2000

This also appears in "Current Protocols in Molecular Biology"

Error-prone PCR (EP-PCR) is the method of choice for introducing random mutations into a defined segment of DNA that is too long to be chemically synthesized as a degenerate sequence (UNIT 8.2A). The 5' and 3' boundaries of the mutated region may be defined by the choice of PCR primers. Accordingly it is possible to mutagenize an entire gene or merely a segment of a gene. The average number of mutations per DNA fragment can be controlled by the number of EP-PCR doublings performed.

The EP-PCR technique described here is based on the protocol of Cadwell and Joyce (1992). This reference also discusses the history of the technique and compares it to other in vitro and in vivo mutagenic methods. EP-PCR takes advantage of the inherently low fidelity of Taq DNA polymerase, which may be further decreased by the addition of Mn²⁺, increasing the Mg²⁺ concentration, and using unequal dNTP concentrations.

After choosing a region of DNA to randomly mutagenize, one must decide on the desired level of mutagenesis that is best suited to the project. If the mutation rate is too low, it may not be possible to find the potentially rare variants of interest. If the mutation rate is too high, nearly all of the resulting library of molecules will carry multiple mutations and may therefore be inactive. The desired extent of mutation depends on the type of activity one is attempting to generate and the number of library members that can be screened. A reasonable approach in many instances is to generate a library such that a few unmutagenized molecules will be present in the collection of screened clones.

Table 2. The fraction of un-mutated DNA templates as a function of template length and number of EP-PCR doublings.

Table 3. The average number of amino acid mutations per open reading frame (ORF) as a function of ORF length and number of EP-PCR doublings.

Table 4. The fraction of ORF's encoding wild-type polypeptide as a function of ORF length and the number of EP-PCR doublings.

This EP-PCR protocol produces all types of substitution mutations, but the distribution is highly non-random. The results we obtained in one experiment are shown in Table 5. It should be noted that in the EP-PCR reaction, both top and bottom DNA strands are equally subject to mutagenesis, so mutations from G to A and from C to T, for example, are combined together.

Table 5. Observation of each type of substitution in a collection of 97 mutations generated by EP-PCR.

Materials

PCR template, 400 bp in length

5' and 3' PCR primers, 100 µM each

dCTP and dTTP, 25 mM each, pH~7

dATP and dGTP, 5 mM each, pH~7

KCl, 2 M

MgCl₂, 200 mM

Tris pH 8.3, 100 mM

MnCl₂, 25 mM

Taq DNA Polymerase, 5U/µL

100 or 600 µl PCR tubes (Sarstedt)

Thermal Cycler (see UNIT 15)

TOPO T/A cloning kit (Invitrogen, Carlsbad, CA)

QIAprep kit (Qiagen)

1. Make up the following PCR reaction mixture on ice.

Concentration

Reagent Amount Stock in PCR reaction

Water 51 µl

Tris pH 8.3 10 µL 100 mM 10 mM

KCl 2.5 µL 2 M 50 mM

Taq DNA Polymerase 1 µl 5U/µL 0.05 U/µL

Total 100µl

1. Place in the thermal cycler and perform about 12 PCR cycles (UNIT 15), or enough to obtain a 1000-fold (10 doublings) increase in the amount of PCR product relative to the input template. The cycling conditions will vary depending on the template and primers, but reasonable starting conditions are: 94° for 1 min, 60° for 1min, 72° for 3 min.

2. Run an ethidium bromide-containing agarose gel to confirm the amount and correct molecular weight of the product (UNIT 2.7).

3. Clone and sequence a sample of the resulting PCR DNA to determine the frequency of mutations in the product. This can be done using the TOPO T/A cloning kit (Invitrogen) and the QIAprep kit (Qiagen). For information on DNA sequencing, see UNIT 7.

Sometimes it is desirable to mutagenize an entire collection of sequences simultaneously. The basic protocol above is appropriate in cases where the starting template is a unique sequence, but the following modifications are recommended when the starting template is itself a library.

The protocol above calls for the EP-PCR reaction to be initiated with a very small amount of template, but this amount may be insufficient to preserve the initial library complexity. To avoid complexity loss before and during the amplification process, one can start with a comparatively large template concentration and perform only four EP-PCR cycles, and then transfer ~10% of the resulting material into a fresh EP-PCR reaction. These "serial transfers" are continued until the desired number of doublings is achieved. One additional advantage of this method is that the progress of the EP-PCR reaction can be monitored throughout the entire procedure, a luxury that is not possible using the standard protocol described above.

The following protocol will give approximately 50 EP-PCR doublings and results in mutations in about 3.5% of the nucleotide positions in the DNA template. However, the actual mutagenic rate may vary with conditions and template.

1. Make up the following EP-PCR reaction mixture on ice.

Concentration

Reagent Amount Stock in PCR reaction

Water 960 µl

Tris pH 8.3 150 µL 100 mM 10 mM

KCl 37.5 µL 2 M 50 mM

Taq DNA Polymerase 1 µl 5U/µL 0.05 U/µL

4. Perform four cycles of EP-PCR amplification. During the final extension at 72°C, place the next tube containing the fresh EP-PCR mixture into the same PCR block. Before the final extension is complete but ensuring that the next tube has reached the extension temperature, transfer 10µl of EP-PCR reaction mixture from the first tube into the second, and then add the following to the second tube and mix:

Concentration

Reagent Amount Stock in PCR reaction

Taq DNA Polymerase 1 µl 5U/µL 0.05 U/µL

Remove the first EP-PCR reaction mixture from the block and store at 4°C.

5. Repeat step four 14 times. Analyze the PCR reaction using agarose gel electrophoresis (UNIT 2.7) after every fourth transfer, and quantitate the bands in successive PCR amplifications.

Before the entire EP-PCR protocol is attempted, it is important to pilot the EP-PCR conditions to ensure that low molecular weight PCR products are not "taking over" the reaction, and that the amplification per cycle is at least 1.7. The optimal PCR amplification conditions may be different from normal PCR amplification performed upon the same library.

This serial transfer approach yields a succession of samples with increasing levels of mutagenesis. If one is uncertain about the optimal level of mutagenesis for a particular application, the samples from different stages of the EP-PCR procedure can be mixed prior to screening or selection.

Cadwell, R. C. and Joyce, G. F. 1992. Randomization of Genes by PCR Mutagenesis. PCR Meth. Appl. 2, 28-33.