Heat‐Activatable Primers for Hot‐Start PCR: Oligonucleotide Synthesis and Basic PCR Setup

Alexandre Lebedev1

1 TriLink BioTechnologies, Inc., San Diego, California
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.35
DOI:  10.1002/0471142700.nc0435s38
Online Posting Date:  September, 2009
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Abstract

2′‐Deoxyribonucleoside‐3′‐O‐(4‐oxotetradec‐1‐yl) phosphoramidites (OXT phosphoramidites) are used to prepare modified oligodeoxyribonucleotide primers containing heat cleavable OXT phosphotriester protecting groups at 3′‐ultimate and penultimate internucleotide linkages. The OXT‐modified primers significantly improve performance of the polymerase chain reaction (PCR) compared to standard DNA primers by substantially reducing or eliminating the accumulation of PCR artifacts such as dimerized primers and misprimed amplicons. Basic protocols for synthesis of OXT‐modified oligonucleotide primers and for performing hot‐start PCR are described. Curr. Protoc. Nucleic Acid Chem. 38:4.35.1‐4.35.17. © 2009 by John Wiley & Sons, Inc.

Keywords: PCR; hot‐start PCR; primer dimer; mispriming; 3′‐phosphoramidite; phosphotriester; oligonucleotide; DNA polymerase

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis and Purification of Oligonucleotides Containing 4‐Oxotetradec‐1‐yl Phosphotriester Internucleotide Linkage(s)
  • Alternate Protocol 1: Desalting of OXT‐Modified Oligonucleotides on SepPak Cartridges Using Water‐Acetonitrile Mixture for Elution
  • Basic Protocol 2: Hot‐Start PCR with OXT‐Modified Oligonucleotide Primers and End‐Point Detection
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis and Purification of Oligonucleotides Containing 4‐Oxotetradec‐1‐yl Phosphotriester Internucleotide Linkage(s)

  Materials
  • 2′‐Deoxyribonucleoside OXT phosphoramidite S.4a‐d
  • Anhydrous acetonitrile, synthesis grade
  • Dichloromethane
  • Molecular sieves, type 3Å, activated
  • Argon gas (dry)
  • Fast deprotecting 2‐cyanoethyl deoxyribonucleoside phosphoramidite monomers (Glen Research)
  • Fast deprotecting OXT deoxyribonucleoside phosphoramidite monomers (Trilink Biotechnologies)
  • Cap A mixture: 5% phenoxyacetic anhydride in THF (Glen Research cat. no. 40‐4212‐52)
  • Controlled pore glass (CPG)
  • 50 mM potassium carbonate solution in methanol (Glen Research)
  • 1 M triethylammonium acetate buffer (TEAA), pH 7.2 (Trilink Biotechnologies)
  • Reversed‐phase C 18 cartridge for oligonucleotide isolation (SepPak, Waters)
  • Washing solution 1: 25 mM TEAA, pH 7.2
  • Water, HPLC grade (Fisher Scientific)
  • 2% trifluoroacetic acid (TFA) solution in water
  • Washing solution 2: 15% acetonitrile in 100 mM TEAA, pH 7.2
  • Washing solution 3: 25% acetonitrile in 100 mM TEAA, pH 7.2
  • DMSO (Sigma‐Aldrich)
  • Mobile phase A1: 50 mM TEAA, pH 7.2
  • Mobile phase B: 100% acetonitrile
  • Mobile phase A2: 50 mM TEAB, pH 8.5
  • 1 M triethylammonium bicarbonate buffer (TEAB), pH 8.5 (Sigma‐Aldrich)
  • DeltaPak 15‐µm C 18 (3.9 × 300–mm) analytical column (Waters)
  • DeltaPak 15‐µm C 18 (19 × 300–mm) preparative column (Waters)
  • 4‐ and 8‐mL screw‐capped glass vials
  • Rotary mixer
  • 15‐ and 50‐mL screw‐cap plastic tubes
  • Speedvac concentrator equipped with condensation trap (e.g., model SC110, Savant)
  • UV‐VIS spectrophotometer (Beckman, model DU640B or equivalent)
  • 1‐, 10‐, 15‐, and 50‐ mL plastic syringes
  • Mass spectrometer (Finnigan MAT LCQ mass spectrometer or equivalent), optional
  • HPLC system with diode array detector (System Gold, Beckman or equivalent), optional
  • Additional reagents and equipment for solid‐phase oligonucleotide synthesis ( appendix 3C) and RP‐HPLC (unit 10.5)

Alternate Protocol 1: Desalting of OXT‐Modified Oligonucleotides on SepPak Cartridges Using Water‐Acetonitrile Mixture for Elution

  • 1.5‐mL plastic tubes

Basic Protocol 2: Hot‐Start PCR with OXT‐Modified Oligonucleotide Primers and End‐Point Detection

  Materials
  • 10× MgCl 2‐free PCR buffer, pH 8.4 (Invitrogen)
  • 50 mM MgCl 2 solution (Invitrogen)
  • 10 mM dNTP mix (Invitrogen)
  • 2.5 U/µL Taq DNA polymerase (native or recombinant)
  • Template DNA, e.g., 1µg/mL bacteriophage λ‐genomic DNA
  • 50 µM standard DNA primer 1 at 50 pmol/µL in water (store at −20°C)
  • 50 µM standard DNA primer 2 at 50 pmol/µL in water (store at −20°C)
  • 200 µM singly OXT‐modified primer 1 at 200 pmol/µL in DMSO (store at −20°C)
  • 200 µM singly OXT‐modified primer 2 at 200 pmol/µL in DMSO (store at −20°C)
  • 200 µM doubly OXT‐modified primer 1 at 200 pmol/µL in DMSO (store at −20°C)
  • 200 µM doubly OXT‐modified primer 2 at 200 pmol/µL in DMSO (store at −20°C)
  • DMSO
  • Mineral oil (optional, Sigma)
  • Water, biology grade or HPLC grade (Fisher Scientific)
  • 50‐bp DNA ladder diluted 1:50 with HPLC water (Invitrogen, cat. no. 10416‐014)
  • 0.2‐mL thin‐walled PCR tubes
  • Microcentrifuge with PCR tubes rotor (Cole Parmer, K‐17310‐00 or equivalent)
  • Automated thermal cycler
  • E‐Gel powerbase v.4 and pre‐cast 4% agarose gel (Invitrogen, cat. no. 6018‐02)
  • Additional reagents and equipment for performing agarose gel electrophoresis (e.g., Voytas, )
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Figures

Videos

Literature Cited

Literature Cited
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