The use of PIPE cloning at TMPC

Technology

Cloning

Summary

Polymerase Incomplete Primer Extension (PIPE) cloning is a ligase free cloning technology developed at the Joint Center for Structural Genomics (1,2). We use PIPE cloning extensively for the initial creation of expression clones. When the clones are made PIPE technology is also used to modify them by deleting or exchanging tag or linker sequences, creating truncation variants, and making point mutations.

Description

The PIPE method depends on the discovery that during the later cycles of normal PCR, as the concentration of various polymerase substrates get used up, a population DNA molecules that are partially single stranded at the 5’-end are generated. By designing complementary 5’-ends that can anneal together provides the means for combining the PCR fragments for either insert cloning or mutagenesis. Figure 1 shows an example how we use PIPE technology to clone inserts into expression vectors. In this example the insert is PCR amplified while appending a sequence encoding the TEV protease cleavage site to the 5’ end of the insert. The vector is PCR amplified using a reverse primer containing the complement of the TEV sequence and a forward primer that deletes the barnase-CAT (Bar-Cat) lethal cassette. The PCR products are simply mixed together, so intermolecular complementation can occur, and transformed into a highly competent E. coli strain. Apparently, the annealed vector and insert gets transformed into the cell and the ends are repaired and ligated in vivo creating a replicating plasmid. The vector template plasmid will not grow by virtue of the Bar-Cat cassette, thus greatly reducing background. If the insert template has the same antibiotic resistance of the expression vector, the insert PCR reaction can be treated with Dpn1 to remove the template after PCR, to eliminate the background caused by the insert template. Figure 2 shows the primer configurations needed to make deletions, insertions, and substitutions of an expression plasmid. Since this type of mutagenesis only requires one PCR reaction and intra-molecular complementation is more efficient than insert cloning. To reduce the background of non-modified template a Dpn1 digestion is routinely done immediately after cycling is complete. We have used this technology to quickly make truncations of mitochondrial-targeted proteins to remove transit sequences. We also used it to delete affinity tags our clones for doing protein-protein interaction studies. We’ve quickly exchange tags and linker sequences and routinely create point mutations using the PIPE technology. PIPE cloning is a cheap and easy technology that has been invaluable in our protein production platform.

Authors: Wrobel RL, Gesior AP, Saunders JM

Figure


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Figure 1. PIPE cloning of ORFs into pEU-His-Flexi with the addition of a TEV protease cleavage site.

Figure 2. Primer configurations for creating deletions, insertions, and substitutions using PIPE technology.

Publication

Klock HE, Koesema EJ, Knuth MW, Lesley SA. Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts. Proteins (2008) May 1;71(2):982-94. PubMed ID: 18004753 | Search SBKB Publications portal

Klock HE, Lesley SA. The Polymerase Incomplete Primer Extension (PIPE) method applied to high-throughput cloning and site-directed mutagenesis. Methods Mol Biol (2009); 498:91-103. PubMed ID: 18988020 | Search SBKB Publications portal

Contact

Brian G. Fox (bgfox@biochem.wisc.edu)

Transmembrane Protein Center

Availability

Email for details

Link




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Last edited:Mon 10 Mar 2014 - 3 years, 3 months ago