Abstract
Touchdown (TD) PCR offers a simple and rapid means to optimize PCRs, increasing specificity, sensitivity and yield, without the need for lengthy optimizations and/or the redesigning of primers. TD-PCR employs an initial annealing temperature above the projected melting temperature (Tm) of the primers being used, then progressively transitions to a lower, more permissive annealing temperature over the course of successive cycles. Any difference in Tm between correct and incorrect annealing will produce an exponential advantage of twofold per cycle. TD-PCR has found wide applicability in standard PCR protocols, including reverse transcriptase-dependent PCR, as well as in the generation of cDNA libraries and single nucleotide polymorphism screening. TD-PCR is particularly useful for templates that are difficult to amplify but can also be standardly used to enhance specificity and product formation. The procedure takes between 90 and 120 min, depending on the template length.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Saiki, R.K. et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–1354 (1985).
Saiki, R.K. et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491 (1988).
Mullis, K. et al. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb. Symp. Quant. Biol. 51 (Part 1): 263–273 (1986).
Kolmodin, L.A. & Birch, D.E. Polymerase chain reaction. Basic principles and routine practice. Methods Mol. Biol. 192, 3–18 (2002).
Kramer, M.F. & Coen, D.M. Enzymatic amplification of DNA by PCR: standard procedures and optimization. Curr. Protoc. Mol. Biol. Chapter 15, Unit 15 1 (2001).
Don, R.H., Cox, P.T., Wainwright, B.J., Baker, K. & Mattick, J.S. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 19, 4008 (1991).
Wu, W.M. et al. Touchdown thermocycling program enables a robust single nucleotide polymorphism typing method based on allele-specific real-time polymerase chain reaction. Anal. Biochem. 339, 290–296 (2005).
Nolan, T., Hands, R.E. & Bustin, S.A. Quantification of mRNA using real-time RT-PCR. Nat. Protoc. 1, 1559–1582 (2006).
Panjkovich, A. & Melo, F. Comparison of different melting temperature calculation methods for short DNA sequences. Bioinformatics 21, 711–722 (2005).
Kibbe, W.A. OligoCalc: an online oligonucleotide properties calculator. Nucleic Acids Res. 35, W43–W46 (2007).
Borer, P.N., Dengler, B., Tinoco Jr. I. & Uhlenbeck, O.C. Stability of ribonucleic acid double-stranded helices. J. Mol. Biol. 86, 843–853 (1974).
Steger, G. Thermal denaturation of double-stranded nucleic acids: prediction of temperatures critical for gradient gel electrophoresis and polymerase chain reaction. Nucleic Acids Res. 22, 2760–2768 (1994).
SantaLucia Jr. J. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc. Natl. Acad. Sci. USA 95, 1460–1465 (1998).
SantaLucia Jr. J., Allawi, H.T. & Seneviratne, P.A. Improved nearest-neighbor parameters for predicting DNA duplex stability. Biochemistry 35, 3555–3562 (1996).
Sugimoto, N., Nakano, S., Yoneyama, M. & Honda, K. Improved thermodynamic parameters and helix initiation factor to predict stability of DNA duplexes. Nucleic Acids Res. 24, 4501–4505 (1996).
Hecker, K.H. & Roux, K.H. High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR. Biotechniques 20, 478–485 (1996).
Zhang, Z. & Martineau, D. Single-tube heminested PCR coupled with 'touchdown' PCR for the analysis of the walleye dermal sarcoma virus env gene. J. Virol. Methods 60, 29–37 (1996).
Rubie, C. et al. Multistep-touchdown vectorette-PCR—a rapid technique for the identification of IVS in genes. Biotechniques 27, 414–416, 418 (1999).
Duckworth, A.W. & Rule, S.A. The use of 'touchdown' polymerase chain reaction increases the sensitivity and specificity of t(11;14)(q13;q32) detection in patients with mantle cell lymphoma. Br. J. Haematol. 121, 952–953 (2003).
De la Horra, C. et al. Comparison of single and touchdown PCR protocols for detecting Pneumocystis jirovecii DNA in paraffin-embedded lung tissue samples. J. Eukaryot. Microbiol. 53 (Suppl 1): S98–S99 (2006).
Fietto, J.L., DeMarco, R. & Verjovski-Almeida, S. Use of degenerate primers and touchdown PCR for construction of cDNA libraries. Biotechniques 32, 1404–1408, 1410–1411 (2002).
Piraee, M. & Vining, L.C. Use of degenerate primers and touchdown PCR to amplify a halogenase gene fragment from Streptomyces venezuelae ISP5230. J. Ind. Microbiol. Biotechnol. 29, 1–5 (2002).
Levano-Garcia, J., Verjovski-Almeida, S. & da Silva, A.C. Mapping transposon insertion sites by touchdown PCR and hybrid degenerate primers. Biotechniques 38, 225–229 (2005).
Kornmann, B., Preitner, N., Rifat, D., Fleury-Olela, F. & Schibler, U. Analysis of circadian liver gene expression by ADDER, a highly sensitive method for the display of differentially expressed mRNAs. Nucleic Acids Res. 29, E51–E51 (2001).
Ding, W., Zou, H., Dai, J. & Duan, Z. Combining restriction digestion and touchdown PCR permits detection of trace isoforms of histamine H3 receptor. Biotechniques 39, 841–845 (2005).
Li, J., Kuang, K., Nielsen, S. & Fischbarg, J. Molecular identification and immunolocalization of the water channel protein aquaporin 1 in CBCECs. Invest. Ophthalmol. Vis. Sci. 40, 1288–1292 (1999).
Ault, G.S., Ryschkewitsch, C.F. & Stoner, G.L. Type-specific amplification of viral DNA using touchdown and hot start PCR. J. Virol. Methods 46, 145–156 (1994).
Roux, K.H. Single-step PCR optimization using touchdown and stepdown PCR programming. Methods Mol. Biol. 192, 31–36 (2002).
Strauss, W.M. Preparation of genomic DNA from mammalian tissue. Curr. Protoc. Mol. Biol., Chapter 2, Unit 2.2 (2001).
Chomczynski, P. & Sacchi, N. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat. Protoc. 1, 581–585 (2006).
Rozen, S. & Skaletsky, H. Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132, 365–386 (2000).
Yuryev, A.E. PCR Primer Design (ed. Yuryev, A.) (Humana Press, Totowa, NJ, 2007).
Brody, J.R., Calhoun, E.S., Gallmeier, E., Creavalle, T.D. & Kern, S.E. Ultra-fast high-resolution agarose electrophoresis of DNA and RNA using low-molarity conductive media. Biotechniques 37, 598, 600, 602 (2004).
Brody, J.R. & Kern, S.E. History and principles of conductive media for standard DNA electrophoresis. Anal. Biochem. 333, 1–13 (2004).
Mokaddas, E., Ahmad, S. & Abal, A.T. Molecular fingerprinting of isoniazid-resistant Mycobacterium tuberculosis isolates from chest diseases hospital in Kuwait. Microbiol. Immunol. 46, 767–771 (2002).
Ahmad, S., Mokaddas, E. & Jaber, A.A. Rapid detection of ethambutol-resistant Mycobacterium tuberculosis strains by PCR-RFLP targeting embB codons 306 and 497 and iniA codon 501 mutations. Mol. Cell. Probes 18, 299–306 (2004).
Ahmad, S. & Mokaddas, E. The occurrence of rare rpoB mutations in rifampicin-resistant clinical Mycobacterium tuberculosis isolates from Kuwait. Int. J. Antimicrob. Agents 26, 205–212 (2005).
Mokaddas, E. & Ahmad, S. Species spectrum of nontuberculous mycobacteria isolated from clinical specimens in Kuwait. Curr. Microbiol. 56, 413–417 (2008).
Harboe, M. et al. Cross-reaction between mammalian cell entry (Mce) proteins of Mycobacterium tuberculosis. Scand. J. Immunol. 56, 580–587 (2002).
Ahmad, S., El-Shazly, S., Mustafa, A.S. & Al-Attiyah, R. Mammalian cell-entry proteins encoded by the mce3 operon of Mycobacterium tuberculosis are expressed during natural infection in humans. Scand. J. Immunol. 60, 382–391 (2004).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Korbie, D., Mattick, J. Touchdown PCR for increased specificity and sensitivity in PCR amplification. Nat Protoc 3, 1452–1456 (2008). https://doi.org/10.1038/nprot.2008.133
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2008.133
This article is cited by
-
Finding stable and closely linked QTLs against spot blotch in different planting dates during the adult stage in barley
Scientific Reports (2024)
-
First report of crown rot on lettuce caused by Phytophthora crassamura in Japan
Journal of General Plant Pathology (2024)
-
TaME-seq2: tagmentation-assisted multiplex PCR enrichment sequencing for viral genomic profiling
Virology Journal (2023)
-
A biological camera that captures and stores images directly into DNA
Nature Communications (2023)
-
Distinct roles of Arabidopsis ORC1 proteins in DNA replication and heterochromatic H3K27me1 deposition
Nature Communications (2023)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.