• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Mar 15, 1994; 91(6): 2216–2220.
PMCID: PMC43341

PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates.

Abstract

A target length limitation to PCR amplification of DNA has been identified and addressed. Concomitantly, the base-pair fidelity, the ability to use PCR products as primers, and the maximum yield of target fragment were increased. These improvements were achieved by the combination of a high level of an exonuclease-free, N-terminal deletion mutant of Taq DNA polymerase, Klentaq1, with a very low level of a thermostable DNA polymerase exhibiting a 3'-exonuclease activity (Pfu, Vent, or Deep Vent). At least 35 kb can be amplified to high yields from 1 ng of lambda DNA template.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.5M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • 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]
  • Lundberg KS, Shoemaker DD, Adams MW, Short JM, Sorge JA, Mathur EJ. High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene. 1991 Dec 1;108(1):1–6. [PubMed]
  • Lawyer FC, Stoffel S, Saiki RK, Chang SY, Landre PA, Abramson RD, Gelfand DH. High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5' to 3' exonuclease activity. PCR Methods Appl. 1993 May;2(4):275–287. [PubMed]
  • Barnes WM. The fidelity of Taq polymerase catalyzing PCR is improved by an N-terminal deletion. Gene. 1992 Mar 1;112(1):29–35. [PubMed]
  • Jeffreys AJ, Wilson V, Neumann R, Keyte J. Amplification of human minisatellites by the polymerase chain reaction: towards DNA fingerprinting of single cells. Nucleic Acids Res. 1988 Dec 9;16(23):10953–10971. [PMC free article] [PubMed]
  • Krishnan BR, Kersulyte D, Brikun I, Berg CM, Berg DE. Direct and crossover PCR amplification to facilitate Tn5supF-based sequencing of lambda phage clones. Nucleic Acids Res. 1991 Nov 25;19(22):6177–6182. [PMC free article] [PubMed]
  • Maga EA, Richardson T. Amplification of a 9.0-kb fragment using PCR. Biotechniques. 1991 Aug;11(2):185–186. [PubMed]
  • Ohler LD, Rose EA. Optimization of long-distance PCR using a transposon-based model system. PCR Methods Appl. 1992 Aug;2(1):51–59. [PubMed]
  • Rychlik W, Spencer WJ, Rhoads RE. Optimization of the annealing temperature for DNA amplification in vitro. Nucleic Acids Res. 1990 Nov 11;18(21):6409–6412. [PMC free article] [PubMed]
  • Kainz P, Schmiedlechner A, Strack HB. In vitro amplification of DNA fragments greater than 10 kb. Anal Biochem. 1992 Apr;202(1):46–49. [PubMed]
  • Chu G, Vollrath D, Davis RW. Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science. 1986 Dec 19;234(4783):1582–1585. [PubMed]
  • Ippen K, Shapiro JA, Beckwith JR. Transposition of the lac region to the gal region of the Escherichia coli chromosome: isolation of lambda-lac transducing bacteriophages. J Bacteriol. 1971 Oct;108(1):5–9. [PMC free article] [PubMed]
  • Shpakovski GV, Akhrem AA, Berlin YuA Structural bases of a long-stretched deletion: completing the lambda plac5 DNA primary structure. Nucleic Acids Res. 1988 Nov 11;16(21):10199–10212. [PMC free article] [PubMed]
  • Tailor R, Tippett J, Gibb G, Pells S, Pike D, Jordan L, Ely S. Identification and characterization of a novel Bacillus thuringiensis delta-endotoxin entomocidal to coleopteran and lepidopteran larvae. Mol Microbiol. 1992 May;6(9):1211–1217. [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]
  • Brewer AC, Marsh PJ, Patient RK. A simplified method for in vivo footprinting using DMS. Nucleic Acids Res. 1990 Sep 25;18(18):5574–5574. [PMC free article] [PubMed]
  • Lindahl T. Instability and decay of the primary structure of DNA. Nature. 1993 Apr 22;362(6422):709–715. [PubMed]
  • Lindahl T, Nyberg B. Rate of depurination of native deoxyribonucleic acid. Biochemistry. 1972 Sep 12;11(19):3610–3618. [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Cited in Books
    Cited in Books
    PubMed Central articles cited in books
  • MedGen
    MedGen
    Related information in MedGen
  • PubMed
    PubMed
    PubMed citations for these articles

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...