Step 1. Generation of U-containing dsDNA template via PCR
This step uses standard PCR to generate the U-containing dsDNA template. One needs to use a polymerase capable of incorporating dUTPs like Taq (NEB) or Phusion U hotstart (Thermo Fisher). In addition, one must used a dNTP mix containing dUTP instead of dTTPs. Finally, the 5' forward primer must be phosphorylated, and the 3' reverse primer is not. Shown below in Table 1.1 is our standard PCR setup for a 1 kB product using NEB Taq polymerase.
Set up PCR reaction according to manufacturer's suggestion, as shown in Table 1.2 below.
Run the thermocycling program for the PCR. Optimize if necessary. This example is for a 1050 bp product (Table 1.3).
Gel purify UTP PCR#1 via any standard kit. Measure the A260 of the purified PCR product and calculate its concentration. Use this as your template for your second round of UTP containing PCR (UTP PCR#2). This step is optional. This PCR is identical to the UTP PCR#1, but used the gel purified PCR#1 as the template. It is useful to scale up the PCR at this point, if the PCR is sufficiently optimized. Typically, we perform 10 x 50uL PCR reactions at this point (Table 1.4). Using UTP PCR#1 as a template is not strictly essential, but this step minimizes wt background in the final library.
Analyze UTP PCR#2 by agarose gel. If only one product band is visible, one can purify the product using a spin column. If multiple bands are visible, purify DNA by gel extraction. Yields ~ 10 ug of purified dU containing PCR product are desirable at this point. Store PCR products at -20C until ready for use.
Step 2. Degrading the Non-Template Strand with Lambda Exonuclease
Continue to prepare the ssDNA U-containing transient template, starting from "U-containing PCR2". Lambda Exonuclease is a 5'->3' endonuclease that prefers 5' phospho dsDNA >> nonphospho DNA and thus will generate ssDNA of the nonphospho strand. Thus, this enzyme will digest the 'non-template' 5'phosphorylated strand. This step involves digestion of the 5'phosphorylated top strand of PCR2 and gel extraction of the undigested bottom strand. Typically, multiple reactions are performed, followed by gel extraction.
Digest 2-3 μg of the purified second PCR2 product from STEP 1 in a reaction with 4 μL of 10X lambda exonuclease buffer, 10 U lambda exonuclease, and water to 40 μL. Incubate at 37°C 1.5 hour. Heat Inactivate 75°C for 10 min.
Typically, do 2-3 40 uL reactions at this point.
Note: NEB mentions in their product notes :
"5’-OH ends are digested 20X slower than 5’-PO4 ends. ssDNA is digested 100X slower than dsDNA". Since our ssDNA is the "bottom" strand, one can pcr a longer piece of DNA (move the 3' reverse primer farther out) or, do the final amplification further inside the template.
Table 2.1 shows a typical lambda exonuclease reaction.
Gel purification of ssDNA.
Pour a 0.7 % agarose gel using SYBR™ Green II RNA Gel Stain for better visibility of ssDNA product. Isolate the ssDNA band and extract from gel using kit (MN, NEB, Zymo kits work well). Elute in buffer without EDTA. Measure A260 and calculate ssDNA concentration. One can use extinction coefficient of 33 ug/ml, or calculate the extinction coefficient from the sequence. Typical yields for our 1 kb product are approximately 25 ng/uL. Store ssDNA at -20C until ready for use.
Test the quality of the ssDNA by performing a Uracil- DNA Glycosylase (UDG) test.
One may skip this test, but if there is no amplified library pcr product after the final PCR, perform this step to assess the ssDNA. In this step, perform a series of UDG (Uracil- DNA Glycosylase) digestions followed by PCR to determine the minimum amount ssDNA needed for the library generation. This step also tests the quality of the ssDNA template. In the presence of UDG, only contaminating wt parent DNA will yield a PCR product. If there is a PCR product in the presence of UDG, re prepare the ssDNA. For this test, prepare a series of dilutions of the ssDNA template, 1:10, 1:100, 1:1000, 1:1000. For each dilution, setup two 10 uL reactions +/- UDG. Incubate these reactions at 37 C for 30'. Next, using 1 uL of the UDG reaction as template, amplify each sample using Taq DNA polymerase in a 50 uL pcr reaction, and run the results on an agarose gel. An example is shown in Table 2.2.
These next three steps can be done in one day: Annealing, Extension & Ligation, and UDG digestion.
Step 3. Anneal Donor primers to ssDNA template
Donor primer design.
We refer to the mixed base primers as our donor or mutagenesis primers. Donor primers contain the desired mutations near the center of their sequences. The mutations can occur anywhere in the gene, as long as the donor primers do not overlap. Multiple donor primers may be used at one time. To date, the greatest number we have tested is six separate donor primers, each containing mutations. These primers must be 5' phosphorylated. They need an annealing temperature of at least 55 C for use in this protocol. These donor primers typically contain 15-20 homologous bases on either side of the mutations. It is not necessary to purify these primers. A python script called MSCS (Mixed Synthesis Codon Selector) has been written to help design primers. To date, the shortest donor primer used is 29 bases, and the longest 68 bases.
Make the DNA library after verifying the ssDNA is of sufficient quality by doing a UDG test.
The donor primers anneal to the "U" containing single stranded (ss) transient template DNA at room temperature. This results in better stoichiometry of a given donor primer because differences in affinity of the mutations do not play as large a role. One also needs a standard 5' forward primer in this annealing step (phosphorylation state unimportant). Each reaction is allowed to continue for 30 minutes for convenience, although shorter times (except for the UDG digestion) would be acceptable. In general, these reactions are performed on a 10 uL scale, but this could be scaled up if desired. The way the protocol is written, the original sample gets diluted about 800X. One could use greater amounts from each step, but keep in mind the dilution effects.
Note: Aliquot and store NEB Taq DNA ligase buffer at -80C and do not reuse after a thaw, as per manufacturer's recommendations.
Lyophilized primers are typically resuspended to a concentration of 100 uM in sterile 10 mM Tris pH 8.5 and stored at -20 C until ready for use.
A typical 10 uL annealing reaction is shown below in Table 3.1. The desired set of donor primers are mixed together to 10 uM. The reagents are mixed, spun down, and incubated at room temperature on the bench (18-20 C) for 30'. In this reaction, the primers are in 1000x molar excess of the ssDNA template. In practice, the amount of ssDNA used as the template is determined by the UDG test in Step 2. These are suggested starting amounts.
Step 4. Extend and Ligate Donor primers
Prepare a 10 uL extension and ligation reaction in a PCR tube. Incubate this reaction at 55C for 30' (again this could probably be shorter, but 30' seems fine). This is shown in Table 4.1. Typically, the next step uses 2.5 uL and the the remainder is stored at -20C.
Step 5. UDG digestion of ssDNA template
This step selectively digests the wt u-containing ssDNA template with UDG.Typically, a 10 uL reaction is performed using 2.5 uL of the extended and ligated sample. An example of the UDG reaction is shown below in Table 5.1. The reaction is incubated for 30' at 37 C. Heat inactivation is not necessary this UDG.
Step 6. Amplification of library via high fidelity DNA polymerase
Typically, 2.5 uL of the above sample is used as a template in a 50 uL PCR reaction using either Taq or Phusion polymerase. Either polymerase should work, but depending on the product, one or the other may be preferable. For example, as Phusion DNA polymerase cannot amplify u-containing templates, using this polymerase would further ensure no wt background, plus it has a higher fidelity. If there is a single amplified product on an agarose gel, the product may be cleaned up using a spin column method. If there are multiple bands, gel purification may be necessary. To verify mutations, the purified product is subjected to Sanger sequencing. Ideally, sequencing primers should be located ~100 base pairs away from the mutated region. The library is now ready for use. For example, if the ends of the pcr product contain restriction sites, the library may be digested with those enzymes, cleaned up and ligated into an appropriate vector.