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Protocolo Cloning PCR

Cloning PCR-Generated DNA Fragment

Many methods exist with which to clone PCR-derived DNA fragments. These methods can be separated into those which require the addition of extra bases to the PCR primers, and those which do not require any added bases. Group I includes methods for the incorporation of appropriate restriction enzyme target sequences on the PCR primers, and the UDG and ligation-independent cloning (LIC) protocols which generate 9-12 base single-stranded ends on the PCR fragment. Group II include the various T/A methods which rely on the terminal extendase activity of some DNA polymerases, and also the blunt-end cloning methods which selectively clone blunt-ended molecules. Group I methods routinely yield extremely high efficiencies, yet due to the ease and lack of expense, Group II methods are often preferred.

Some of the older polymerases commonly used for PCR have different and quite specific extendase activities. This characteristic will decrease cloning efficiencies associated with the T/A cloning methods. Therefore, unless it is known which base is preferentially extended onto the end of completed PCR molecules, blunt-ended cloning should be used (see protocol 2 below). Protocol 1 (PCR-insert polishing) should be used for removing extended bases on insert DNA or in cases where the extendase activity of the polymerase used to generate the PCR insert is not known.Pfu DNA polymerase (Stratagene, La Jolla, CA) is preferentially used in this protocol because it has the ability to remove extended bases (by its 3' to 5' exonuclease activity) and is active only at increased (>65 C) temperature. At the temperature used for ligation (22 C), the polishing enzyme is inactive and therefore does not need to be removed prior to a ligation treatment.

Protocol 2, PCR cloning, increases the yield and efficiency of blunt-ended cloning by incorporating a unit excess of restriction enzyme (compared to the units of T4 DNA ligase) in the ligation reaction. The added restriction enzyme serves to keep an excess of linearized vector in solution by restriction of plasmids that religate intramolecularly. Intermolecular ligation with an insert destroys the endonuclease target site. In addition, a blue-white phenotypic color selection is used to differentiate molecules that contain an insert. Although any blunt-ended restriction enzyme for which the plasmid contains a single site can be used for this procedure, it is preferable to use a rare-target site enzyme. The commercially available restriction enzyme SrfI (Roche) has been successfully used.

Protocol 1. PCR Insert Polishing

Insert polishing is used to remove extended bases from PCR-generated DNA fragments. In general, this will result in an increased amount of blunt-ended DNA molecules available for the cloning reaction.
  • PCR insert (10-500 ng) is added to a 10 ul reaction mix containing 1 ul of Pfu DNA polymerase (2.5 U/ ul, Stratagene), 1x Pfu polymerase buffer, and 1 mM dNTP (0.25 mM each nucleoside triphosphate).
  • A mineral oil overlay is added and the reaction is incubated for 30 minutes at 72 C.
  • A 1-2 ul aliquot can be used in the PCR cloning protocol (below).

Protocol 2. PCR Cloning

  • Restriction-enzyme (Srf ) digested plasmid template DNA (approximately 10 ng) is added to a 10 ul reaction mix containing 0.5 mM ATP, 1x reaction buffer (0.1 M Potassium Acetate, 25 mM Tris-Acetate, pH 7.6, 10 mM Magnesium Acetate, 0.5 mM 2-mercaptoethanol), 2-4 ul PCR-generated insert (see also polishing reaction above), 1 ul of restriction enzyme (Srf I, 5 U/ ul) and 1 ul of T4 DNA ligase (4 U/ ul).
  • The reaction is incubated at room temperature for 1-2 hours.
  • 2 ul is removed and used to transform E. coli.