Abstract
Polymerase chain reaction (PCR) is an oft-used preparatory technique in amplifying specific DNA regions for downstream analysis. The size of an amplicon was initially limited by errors in nucleotide polymerization and template deterioration during thermal cycling. A variant of PCR, designated long-range PCR, was devised to counter these drawbacks and enable the amplification of large fragments exceeding a few kb. In this chapter we describe a protocol for long-range PCR, which we have adopted to obtain products of 6.6, 7.2, 13, and 20 kb from human genomic DNA samples.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT et al (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239(4839):487–491
Ou CY, Kwok S, Mitchell SW, Mack DH, Sninsky JJ, Krebs JW et al (1988) DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells. Science 239(4837):295–297
Barnes WM (1994) PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. Proc Natl Acad Sci U S A 91(6):2216–2220
Cheng S, Fockler C, Barnes WM, Higuchi R (1994) Effective amplification of long targets from cloned inserts and human genomic DNA. Proc Natl Acad Sci U S A 91(12):5695–5699
Cheng S, Chen Y, Monforte JA, Higuchi R, Higuchi R, Van Houten B (1995) Template integrity is essential for PCR amplification of 20- to 30-kb sequences from genomic DNA. PCR Methods Appl 4(5):294–298
Fukami T, Nakajima M, Sakai H, McLeod HL, Yokoi T (2006) CYP2A7 polymorphic alleles confound the genotyping of CYP2A6*4A allele. Pharmacogenomics J 6(6):401–412
National Center for Biotechnology Information (2013) The NCBI handbook [Internet], 2nd edn. National Center for Biotechnology Information, Bethesda, MD. http://www.ncbi.nlm.nih.gov/books/NBK143764/
Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M et al (2012) Primer3 – new capabilities and interfaces. Nucleic Acids Res 40(15):e115
Koressaar T, Remm M (2007) Enhancements and modifications of primer design program Primer3. Bioinformatics 23(10):1289–1291
Cone RW, Fairfax MR (1993) Protocol for ultraviolet irradiation of surfaces to reduce PCR contamination. PCR Methods Appl 3(3):S15–S17
Fox JC, Ait-Khaled M, Webster A, Emery VC (1991) Eliminating PCR contamination: is UV irradiation the answer? J Virol Method 33(3):375–382
Devor EJ, Behlke MA (2004) Oligonucleotide yield, resuspension, and storage. Integrated DNA technologies. https://www.idtdna.com/pages/decoded/decoded-articles/core-concepts/decoded/2012/10/08/dna-oligonucleotide-resuspension-and-storage. Accessed 15 Jun 2016.
Barbas CF, Burton DR, Scott JK, Silverman GJ (2007) Quantitation of DNA and RNA. CSH Protoc. doi:10.1101/pdb.ip47
Röder B, Frühwirth K, Vogl C, Wagner M, Rossmanith P (2010) Impact of long-term storage on stability of standard DNA for nucleic acid-based methods. J Clin Microbiol 48(11):4260–4262
Frackman S, Kobs G, Simpson D, Storts D (1998) Betaine and DMSO: enhancing agents for PCR. Promega Notes 65(27-29):27–29
Wright GE, Niehaus DJ, Drögemöller BI, Koen L, Gaedigk A, Warnich L (2010) Elucidation of CYP2D6 genetic diversity in a unique African population: implications for the future application of pharmacogenetics in the Xhosa population. Ann Hum Genet 74(4):340–350
SantaLucia J Jr (1998) A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci U S A 95(4):1460–1465
Ignatov KB, Kramarov VM (2009) DNA ligases from thermophilic bacteria enhance PCR amplification of long DNA sequences. Biochemistry (Mosc) 74(5):557–561
Chua EW, Miller AL, Kennedy MA (2015) Choice of PCR microtube can impact on the success of long-range PCRs. Anal Biochem 477:115–117
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Chua, E.W., Maggo, S., Kennedy, M.A. (2017). Long Fragment Polymerase Chain Reaction. In: Domingues, L. (eds) PCR. Methods in Molecular Biology, vol 1620. Springer, New York, NY. https://doi.org/10.1007/978-1-4939-7060-5_3
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7060-5_3
Published:
Publisher Name: Springer, New York, NY
Print ISBN: 978-1-4939-7059-9
Online ISBN: 978-1-4939-7060-5
eBook Packages: Springer Protocols