Heat‐Activatable Primers for Hot‐Start PCR and Hot‐Start One‐Step RT‐PCR: Endpoint and Real‐Time Experiments

Elena Hidalgo Ashrafi1, Natasha Paul1

1 TriLink BioTechnologies, Inc., San Diego, California
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 15.9
DOI:  10.1002/0471142727.mb1509s88
Online Posting Date:  October, 2009
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Abstract

Hot‐start PCR is a technique that improves PCR performance by reducing nonspecific amplification during the initial setup stages of the PCR. This unit describes hot‐start PCR protocols which utilize primers containing temperature‐sensitive modifications. The introduction of 4‐oxo‐tetradecyl (OXT) phosphotriester groups onto the 3′ end of the primer allows for primer‐based hot‐start PCR that is amenable for use in a number of PCR‐based applications. The protocols described in this unit utilize OXT‐modified primers in applications such as standard thermal cycling PCR, fast thermal cycling PCR, multiplex PCR, and one‐step reverse‐transcription PCR. This method is also advantageous for instances where improved PCR specificity is desired and a hot‐start polymerase suitable for your application is not available. Curr. Protoc. Mol. Biol. 88:15.9.1‐15.9.15. © 2009 by John Wiley & Sons, Inc.

Keywords: PCR; Hot Start PCR; primer dimer; mispriming; thermal cycler; DNA polymerase; dNTPs; multiplex; fast‐cycling PCR; RT‐PCR; real‐time PCR

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Primer‐Based Hot‐Start PCR Using Standard Thermal Cycling
  • Basic Protocol 2: Primer‐Based Hot‐Start One‐Step Reverse‐Transcription PCR
  • Alternate Protocol 1: Primer‐Based Hot‐Start PCR with Real‐Time Detection
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Primer‐Based Hot‐Start PCR Using Standard Thermal Cycling

  Materials
  • 10× PCR buffer: 200 mM Tris⋅Cl/500 mM KCl, pH 8.3 at 25°C (available in prepared form from Invitrogen)
  • 1 M KCl (for multiplex PCR; see “Note” below)
  • 50 mM MgCl 2 stock (Invitrogen)
  • 10 mM dNTP mixture: 10 mM each of dATP, dCTP, dGTP, and dTTP (New England Biolabs)
  • 5 U/µl Taq DNA polymerase (Invitrogen)
  • CleanAmp Primers (available made‐to‐order through custom oligonucleotide synthesis service of TriLink BioTechnologies); alternatively, purchase CleanAmp phosphoramidites (Glen Research or TriLink BioTechnologies) and synthesize CleanAmp Primers in‐house using solid‐phase oligonucleotide synthesis (see http://www.trilinkbiotech.com/cleanamp/Amidite_procedure.pdf and Lebedev, )
  • Template DNA (10 pg/µl to 1 µg/µl recommended concentration)
  • Thin‐walled PCR tubes
  • Automated thermal cycler
  • UV transilluminator
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5)
NOTE: In multiplex PCR, two or more primer pairs are introduced into the reaction. Although the upper limit for how many CleanAmp Primer pairs can be introduced into a multiplex reaction has yet to be determined, reactions containing up to nine primer pairs have been demonstrated (Shum and Paul, ).NOTE: When performing multiplex PCR, for optimal results, titrate KCl to between 70 and 100 mM final concentration (Henegariu et al., ), being sure to take into account the different amounts of KCl that various PCR buffers contain (most, including the 1× concentration of the buffer described here, contain ∼50 mM KCl).

Basic Protocol 2: Primer‐Based Hot‐Start One‐Step Reverse‐Transcription PCR

  Materials
  • 10× PCR buffer: 200 mM Tris⋅Cl/500 mM KCl, pH 8.3 at 25°C (available in prepared form from Invitrogen)
  • 1 M KCl (for multiplex PCR; see “Note: below)
  • 50 mM MgCl 2 stock (Invitrogen)
  • 10 mM dNTP mixture: 10 mM each of dATP, dCTP, dGTP, and dTTP (New England Biolabs)
  • 50 µM standard poly(dT) primer or random primer
  • 40 U/µl RNase inhibitor (Ambion)
  • 200 U/µl M‐MLV Reverse Transcriptase (Invitrogen) or 200 U/µl SuperScript III Reverse Transcriptase (Invitrogen)
  • 5 U/µl Taq DNA polymerase (Invitrogen)
  • CleanAmp Precision Primers (TriLink BioTechnologies, Inc.): forward and reverse
  • Template: 0.1 to 1 µg/µg total RNA or poly(A) RNA sample
  • Thin‐walled PCR tubes
  • Automated thermal cycler
  • UV transilluminator
  • Additional reagents and equipment for RT‐PCR (unit 15.5) and agarose gel electrophoresis (unit 2.5)
NOTE: When adapting this protocol from single‐plex to multiplex one‐step RT‐PCR, the units of Taq DNA polymerase can be increased up to four times to increase amplicon yield without any additional changes to the reaction setup. Using the buffer conditions described in Table 15.9.2, there is no need to increase the MgCl 2 concentration when the units of Taq are quadrupled. When increasing the number of targets to more than three, the use of a reverse transcriptase that is active at higher temperatures, such as SuperScript III RT, can improve the specificity of the reaction.
Table 5.9.2   MaterialsMaster Mix Reagents for One‐Step RT‐PCR for Reactions with a Final Volume of 50 µl

Component Final concentration Volume for 1 reaction
10× PCR buffer 5 µl
Magnesium chloride (50 mM) 1.5 mM 1.5 µl
dNTP mixture (10 mM of each) 0.16 mM 0.8 µl
Poly(dT) or random primer (50 µM) 1 µM 1 µl
RNase inhibitor (Variable U/µl) 0.1 U/µl Variable
Reverse transcriptase (Variable U/µl) c 1 U/µl Variable
DNA polymerase (Variable U/µl) 0.0125 U/µl Variable
CleanAmp Precision Forward Primer 0.1‐0.5 µM d Variable
CleanAmp Precision Reverse Primer 0.1‐0.5 µM d Variable
Sterile deionized water Up to 49 µl
Total volume (µl) 49 µl

 cM‐MLV‐RT or SuperScript III RT. If other enzyme is used, follow the manufacturer's recommendations.
 dIn initial experiments, it is recommended to perform a titration of primer concentration for optimal performance. Note that the optimal concentration for CleanAmp Primers is not necessarily the same as for unmodified primers and is often higher.

Alternate Protocol 1: Primer‐Based Hot‐Start PCR with Real‐Time Detection

  • For SYBR Green detection:
    • 10,000× SYBR Green I nucleic acid stain (Invitrogen)
    • Passive reference: 1 mM ROX dye (Stratagene)
  • For TaqMan Probe detection:
    • TaqMan Probe (see unit 15.8 for probe design)
    • Passive reference: 1 mM ROX dye
  • Thermal cycler capable of real‐time detection
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Figures

Videos

Literature Cited

   Ankenbauer, W., Heindl, D., Laue, F., and Huber, N. 2003. Composition and method for Hot Start nucleic acid amplification. U.S. Application No. US2003119150(A1).
   Barnes, W.M. and Rowlyk, K.R. 2002. Magnesium precipitate hot start method for PCR. Mol. Cell. Probes 16:167‐171.
   Birch, D.E., Laird, W.J., and Zoccoli, A. 1998. Nucleic acid amplification using a reversibly inactivated thermostable enzyme. U.S. Patent 5677152.
   Bonner, A.G. 2003. Reversible chemical modification of nucleic acids and improved method for nucleic acid hybridization. U.S. Patent No. US2003162199(A1).
   Borns, M. 2007. Hot start polymerase reaction using a thermolabile blocker. U.S. Application No. US2007009922(A1).
   Chou, Q., Russell, M., Birch, D.E., Raymond, J., and Bloch, W. 1992. Prevention of pre‐PCR mis‐priming and primer dimerization improves low‐copy‐number amplifications. Nucleic Acids Res. 20:1717‐1723.
   Eastlund, E. and Mueller, E. 2001. Hot Start RT‐PCR results in improved performance of the enhanced avian RT‐PCR system. LifeScience Quarterly 2:2‐5.
   Fan, X.Y., Lu, G.Z., Wu, L.N., Chen, J.H., Xu, W.Q., Zhao, C.N., and Guo, S.Q. 2006. A modified single‐tube one‐step product‐enhanced reverse transcriptase (mSTOS‐PERT) assay with heparin as DNA polymerase inhibitor for specific detection of RTase activity. J. Clin. Virol. 37:305‐312.
   Henegariu, O., Heerema, N.A., Dlouhy, S.R., Vance, G.H., and Vogt, P.H. 1997. Multiplex PCR: Critical parameters and step‐by‐step protocol. BioTechniques 23:504‐511.
   Kaboev, O.K., Luchkina, L.A., Tret'iakov, A.N., and Bahrmand, A.R. 2000. PCR hot start using primers with the structure of molecular beacons (hairpin‐like structure). Nucleic Acids Res. 28:E94.
   Koukhareva, I., Haoqiang, H., Yee, J., Shum, J., Paul, N., Hogrefe, R.I., and Lebedev, A.V. 2008. Heat activatable 3′‐modified dNTPs: Synthesis and application for hot start PCR. Nucleic Acids Symp. Ser. (Oxf) 52:259‐260.
   Kubu, C.J., Muller‐Greven, J.C., and Moffett, R.B. 2008. Novel hot start nucleic acid amplification. U.S. Application No. US2008138878(A1).
   Laird, W.J. and Niemiec, J.T. 2004. Amplification using modified primers. U.S. Patent No. 6794142.
   Lebedev, A. 2009. Heat‐activatable primers for Hot Start PCR: Oligonucleotide synthesis and basic PCR set‐up. Curr. Protoc. Nucleic Acid Chem. 38:4.35.1‐4.35.10.
   Lebedev, A.V., Paul, N., Yee, J., Timoshchuk, V.A., Shum, J., Miyagi, K., Kellum, J., Hogrefe, R.I., and Zon, G. 2008. Hot start PCR with heat‐activatable primers: A novel approach for improved PCR performance. Nucleic Acids Res. 36:e131.
   Mizuguchi, H., Nakatsuji, M., Fujiwara, S., Takagi, M., and Imanaka, T. 1999. Characterization and application to hot start PCR of neutralizing monoclonal antibodies against KOD DNA polymerase. J. Biochem. (Tokyo) 126:762‐768.
   Moretti, T., Koons, B., and Budowle, B. 1998. Enhancement of PCR amplification yield and specificity using AmpliTaq Gold DNA polymerase. BioTechniques 25:716‐722.
   Sears, J.F. and Khan, A.S. 2003. Single‐tube fluorescent product‐enhanced reverse transcriptase assay with Ampliwax (STF‐PERT) for retrovirus quantitation. J. Virol. Methods 108:139‐142.
   Setterquist, R.A. and Smith, K.G. 1996. Ready to use encapsulated PCR reagents. Nucleic Acids Res. 24:1580‐1581.
   Shum, J. and Paul, N. 2009. Chemically modified primers for improved multiplex polymerase chain reaction. Anal. Biochem. 388:266‐272.
   Tanzer, L.R., Hu, Y., Cripe, L., and Moore, R.E. 1999. A hot‐start reverse transcription‐polymerase chain reaction protocol that initiates multiple analyses simultaneously. Anal Biochem 273:307‐310.
   Ullman, E.F., Lishanski, A., and Kurn, N. 2002. Method for polynucleotide amplification. U.S. Patent No. 6482590(B1).
   Wacker, M.J., and Godard, M.P. 2005. Analysis of one‐step and two‐step Real‐Time RT‐PCR using SuperScript III. J. Biomol. Tech. 16:266‐271.
   Wagner, A., Blackstone, N., Cartwright, P., Dick, M., Misof, B., Snow, P., Wagner, G.P., Bartels, J., Murtha, M., and Pendleton, J. 1994. Surveys of gene families using polymerase reaction: PCR selection and PCR drift. Syst. Biol. 43:250‐261.
Internet Resources
  http://info.med.yale.edu/genetics/ward/tavi/Trblesht.html
  Web site for Multiplex PCR optimization resources.
  http://dyes.gene‐quantification.info/
  Web site for real‐time detection technologies.
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