DNA Shuffling

Materials and Reagents

Fragmentation reaction

In 100 μL combine:

  • 3 μg of the DNA substrate(s)

  • 0.3 units of DNase I (Promega)

  • 10 x RQ1 DNaseI

Primerless PCR mix

  • 10-30 ng/μL DNA

  • 2 μL 10 mM dNTPs

  • 10 x Taq Buffer (2.2 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl, pH 9.0, 0.1% Triton X-100)

  • 2.5 U Taq Polymerase

  • 100 μL total reaction volume

Procedure

Fragmentation

  1. PCR amplify the genes of interest, and gel-purify to remove template and primers.

  2. Incubate for 15-20 minutes at 20 Celsius.

  3. Add to the reaction with RQ1 Stop Buffer.

  4. Incubate at 65 Celsius for 10 minutes.

  5. Purify 50-200 base pair fragments by electrophoresis onto a DE81 paper.

  6. Elute with 1 M NaCl.

  7. Purify DNA by ethanol precipitation.

Primerless PCR

Set up PCR thermocycler:

  • 45 x 95 Celsius for 30s

  • 1 x 50 Celsius for 30s

  • 1 x 72 Celsius for 30s

  • 1 x 72 Celsius for 10 min

Follow-up

  1. Dilute primerless PCR reaction.

  2. Dilute 1:40 into typical PCR mixture, with store-bought Taq Polymerase Buffer and Taq Polymerase.

Notes and Cautions

  • Alternatively, for larger fragments (e.g., >80 base pairs), the appropriately sized bands can be excised from the 2% gel and gel-purified using a commercial kit.

  • Use of Pfu, or homemade Taq buffer (as above in step 3), appears to give an unnecessarily large amount of frame-shifts.

  • The paper, “Optimization of DNA shuffling for high-fidelity recombination”, provides useful tips for how to control the error-rate.

  • For example, using Mn(II) instead of Mg(II) during the DNase I fragmentation step improves fidelity 3-fold (10x reaction buffer: 500 mM Tris-HCl pH 7.4, 100 mM MnCl2).

  • Also, use of higher fidelity polymerases (they use Pfu) for both the original PCR amplification, primerless amplification, and standard primer amplification, can improve fidelity.

  • Additionally, obtaining the fragments to be DNaseI treated from restriction digest of a plasmid can decrease the errors.

  • Protocol adopted from Stemmer, PNAS, 1994.