DNA Cloning and Assembly Methods pp 37-48

Part of the Methods in Molecular Biology book series (MIMB, volume 1116) | Cite as

Quick and Clean Cloning

  • Frank Thieme
  • Sylvestre Marillonnet
Protocol

Abstract

Identification of unknown sequences that flank known sequences of interest requires PCR amplification of DNA fragments that contain the junction between the known and unknown flanking sequences. Since amplified products often contain a mixture of specific and nonspecific products, the quick and clean (QC) cloning procedure was developed to clone specific products only. QC cloning is a ligation-independent cloning procedure that relies on the exonuclease activity of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. A specific feature of QC cloning is the use of vectors that contain a sequence called catching sequence that allows cloning specific products only. QC cloning is performed by a one-pot incubation of insert and vector in the presence of T4 DNA polymerase at room temperature for 10 min followed by direct transformation of the incubation mix in chemo-competent Escherichia coli cells.

Key words

Ligation-independent cloning Flanking sequences Specific products Genome walking Exonuclease digestion 

References

  1. 1.
    Frohman MA, Dush MK, Martin GR (1988) Rapid production of full-length cDNAs from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 85:8998–9002PubMedCrossRefGoogle Scholar
  2. 2.
    Mueller PR, Wold B (1989) In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science 246:780–786PubMedCrossRefGoogle Scholar
  3. 3.
    Riley J, Butler R, Ogilvie D et al (1990) A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones. Nucleic Acids Res 18:2887–2890PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Rosenthal A, Jones DS (1990) Genomic walking and sequencing by oligo-cassette mediated polymerase chain reaction. Nucleic Acids Res 18:3095–3096PubMedCentralPubMedCrossRefGoogle Scholar
  5. 5.
    Liu YG, Whittier RF (1995) Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking. Genomics 25:674–681PubMedCrossRefGoogle Scholar
  6. 6.
    Tonooka Y, Fujishima M (2009) Comparison and critical evaluation of PCR-mediated methods to walk along the sequence of genomic DNA. Appl Microbiol Biotechnol 85:37–43PubMedCrossRefGoogle Scholar
  7. 7.
    Thieme F, Engler C, Kandzia R et al (2011) Quick and clean cloning: a ligation-independent cloning strategy for selective cloning of specific PCR products from non-specific mixes. PLoS One 6:e20556PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Aslanidis C, de Jong PJ (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res 18:6069–6074PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Yang YS, Watson WJ, Tucker PW et al (1993) Construction of recombinant DNA by exonuclease recession. Nucleic Acids Res 21:1889–1893PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Li MZ, Elledge SJ (2007) Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nat Methods 4:251–256PubMedCrossRefGoogle Scholar
  11. 11.
    Bendandi M, Marillonnet S, Kandzia R et al (2010) Rapid, high-yield production in plants of individualized idiotype vaccines for non-Hodgkin’s lymphoma. Ann Oncol 21:2420–2427PubMedCrossRefGoogle Scholar
  12. 12.
    Aslanidis C, de Jong PJ, Schmitz G (1994) Minimal length requirement of the single-stranded tails for ligation-independent cloning (LIC) of PCR products. PCR Methods Appl 4:172–177PubMedCrossRefGoogle Scholar
  13. 13.
    Engler C, Kandzia R, Marillonnet S (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS One 3:e3647PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Frank Thieme
    • 1
  • Sylvestre Marillonnet
    • 2
  1. 1.Icon Genetic GmbHHalleGermany
  2. 2.Department of Cell and Metabolic BiologyLeibniz-Institut für PflanzenbiochemieHalleGermany

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