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Nucleic Acids Res. Oct 25, 1990; 18(20): 6069–6074.
PMCID: PMC332407

Ligation-independent cloning of PCR products (LIC-PCR).

Abstract

A new procedure has been developed for the efficient cloning of complex PCR mixtures, resulting in libraries exclusively consisting of recombinant clones. Recombinants are generated between PCR products and a PCR-amplified plasmid vector. The procedure does not require the use of restriction enzymes, T4 DNA ligase or alkaline phosphatase. The 5'-ends of the primers used to generate the cloneable PCR fragments contain an additional 12 nucleotide (nt) sequence lacking dCMP. As a result, the amplification products include 12-nt sequences lacking dGMP at their 3'-ends. The 3'-terminal sequence can be removed by the action of the (3'----5') exonuclease activity of T4 DNA polymerase in the presence of dGTP, leading to fragments with 5'-extending single-stranded (ss) tails of a defined sequence and length. Similarly, the entire plasmid vector is amplified with primers homologous to sequences in the multiple cloning site. The vector oligos have additional 12-nt tails complementary to the tails used for fragment amplification, permitting the creation of ss-ends with T4 DNA polymerase in the presence of dCTP. Circularization can occur between vector molecules and PCR fragments as mediated by the 12-nt cohesive ends, but not in mixtures lacking insert fragments. The resulting circular recombinant molecules do not require in vitro ligation for efficient bacterial transformation. We have applied the procedure for the cloning of inter-ALU fragments from hybrid cell-lines and human cosmid clones.

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Selected References

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  • Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. [PubMed]
  • Lüdecke HJ, Senger G, Claussen U, Horsthemke B. Cloning defined regions of the human genome by microdissection of banded chromosomes and enzymatic amplification. Nature. 1989 Mar 23;338(6213):348–350. [PubMed]
  • Johnson DH. Molecular cloning of DNA from specific chromosomal regions by microdissection and sequence-independent amplification of DNA. Genomics. 1990 Feb;6(2):243–251. [PubMed]
  • Brooks-Wilson AR, Goodfellow PN, Povey S, Nevanlinna HA, de Jong PJ, Goodfellow PJ. Rapid cloning and characterization of new chromosome 10 DNA markers by Alu element-mediated PCR. Genomics. 1990 Aug;7(4):614–620. [PubMed]
  • Cotter FE, Hampton GM, Nasipuri S, Bodmer WF, Young BD. Rapid isolation of human chromosome-specific DNA probes from a somatic cell hybrid. Genomics. 1990 Jun;7(2):257–263. [PubMed]
  • Clark JM. Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 1988 Oct 25;16(20):9677–9686. [PMC free article] [PubMed]
  • Hemsley A, Arnheim N, Toney MD, Cortopassi G, Galas DJ. A simple method for site-directed mutagenesis using the polymerase chain reaction. Nucleic Acids Res. 1989 Aug 25;17(16):6545–6551. [PMC free article] [PubMed]
  • Nelson DL, Ledbetter SA, Corbo L, Victoria MF, Ramírez-Solis R, Webster TD, Ledbetter DH, Caskey CT. Alu polymerase chain reaction: a method for rapid isolation of human-specific sequences from complex DNA sources. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6686–6690. [PMC free article] [PubMed]
  • Jelinek WR, Schmid CW. Repetitive sequences in eukaryotic DNA and their expression. Annu Rev Biochem. 1982;51:813–844. [PubMed]
  • Jurka J, Smith T. A fundamental division in the Alu family of repeated sequences. Proc Natl Acad Sci U S A. 1988 Jul;85(13):4775–4778. [PMC free article] [PubMed]
  • Kariya Y, Kato K, Hayashizaki Y, Himeno S, Tarui S, Matsubara K. Revision of consensus sequence of human Alu repeats--a review. Gene. 1987;53(1):1–10. [PubMed]
  • Conley EC, Saunders JR. Recombination-dependent recircularization of linearized pBR322 plasmid DNA following transformation of Escherichia coli. Mol Gen Genet. 1984;194(1-2):211–218. [PubMed]
  • Vieira J, Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. [PubMed]
  • Stallings RL, Olson E, Strauss AW, Thompson LH, Bachinski LL, Siciliano MJ. Human creatine kinase genes on chromosomes 15 and 19, and proximity of the gene for the muscle form to the genes for apolipoprotein C2 and excision repair. Am J Hum Genet. 1988 Aug;43(2):144–151. [PMC free article] [PubMed]
  • Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. [PubMed]
  • Goffin C, Bailly V, Verly WG. Nicks 3' or 5' to AP sites or to mispaired bases, and one-nucleotide gaps can be sealed by T4 DNA ligase. Nucleic Acids Res. 1987 Nov 11;15(21):8755–8771. [PMC free article] [PubMed]
  • Wiaderkiewicz R, Ruiz-Carrillo A. Mismatch and blunt to protruding-end joining by DNA ligases. Nucleic Acids Res. 1987 Oct 12;15(19):7831–7848. [PMC free article] [PubMed]
  • Wu DY, Wallace RB. Specificity of the nick-closing activity of bacteriophage T4 DNA ligase. Gene. 1989;76(2):245–254. [PubMed]
  • Scharf SJ, Horn GT, Erlich HA. Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science. 1986 Sep 5;233(4768):1076–1078. [PubMed]
  • Shuldiner AR, Scott LA, Roth J. PCR-induced (ligase-free) subcloning: a rapid reliable method to subclone polymerase chain reaction (PCR) products. Nucleic Acids Res. 1990 Apr 11;18(7):1920–1920. [PMC free article] [PubMed]
  • Jones DH, Sakamoto K, Vorce RL, Howard BH. DNA mutagenesis and recombination. Nature. 1990 Apr 19;344(6268):793–794. [PubMed]

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