Gene Assembly from Chip‐Synthesized Oligonucleotides

Nikolai Eroshenko1, Sriram Kosuri1, Adam H. Marblestone2, Nicholas Conway3, George M. Church3

1 These authors contributed equally to this work, 2 Harvard Biophysics Program, Cambridge, Massachusetts, 3 Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch110190
Online Posting Date:  March, 2012
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Abstract

De novo synthesis of long double‐stranded DNA constructs has a myriad of applications in biology and biological engineering. However, its widespread adoption has been hindered by high costs. Cost can be significantly reduced by using oligonucleotides synthesized on high‐density DNA chips. However, most methods for using off‐chip DNA for gene synthesis have failed to scale due to the high error rates, low yields, and high chemical complexity of the chip‐synthesized oligonucleotides. We have recently demonstrated that some commercial DNA chip manufacturers have improved error rates, and that the issues of chemical complexity and low yields can be solved by using barcoded primers to accurately and efficiently amplify subpools of oligonucleotides. This unit includes protocols for computationally designing the DNA chip, amplifying the oligonucleotide subpools, and assembling 500‐ to 800‐bp constructs. Curr. Protoc. Chem. Biol. 4:1‐17 © 2012 by John Wiley & Sons, Inc.

Keywords: oligonucleotide; gene synthesis; nucleic acids; synthetic biology

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Oligonucleotide Design and Synthesis
  • Basic Protocol 2: PCR Amplification of Oligonucleotide Subpools
  • Basic Protocol 3: Enzymatic Removal of Priming Sites
  • Basic Protocol 4: Assembly of Processed Subpools into Full‐Length Constructs
  • Support Protocol 1: Gel‐Stab PCR
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Oligonucleotide Design and Synthesis

  Materials
  • Computer with a Web browser
  • List of sequences to be built (in FASTA format)

Basic Protocol 2: PCR Amplification of Oligonucleotide Subpools

  Materials
  • Array‐synthesized library
  • 100× TE buffer (Sigma‐Aldirch, cat. no. T9285)
  • Plate subpool‐specific primers ( protocol 1)
  • Assembly subpool‐specific primers ( protocol 1)
  • UltraPure DNase/RNase‐free distilled water (Invitrogen, cat. no. 10977‐023)
  • Phusion high‐fidelity DNA polymerase (New England Biolabs, cat. no. M0530L) containing:
    • 5× Phusion HF Reaction buffer
  • dNTP solution mix (Enzymatics, cat. no. N205L)
  • QIAquick 96 PCR purification kit (QIAGEN, cat. no. 28183)
  • EB buffer (10 mM Tris⋅Cl, pH 8.5)
  • Galaxy microcentrifuge with 1.5‐ml, 2.0‐ml, and 0.2‐ml tube adapters (VWR, cat. no. 37000‐700)
  • Vortex mixer (VWR International, cat. no. 58816‐121)
  • 0.2‐ml PCR tubes with flat caps (BioRad, cat. no. TFI‐0201)
  • Microseal 96‐well skirted low‐profile PCR plates (BioRad, cat. no. MSP‐9601)
  • 96‐Reaction thermal cycler (BioRad, cat. no. 186‐1096)
  • Microseal ‘F’ Sealing Foil (BioRad, cat. no. MSF‐1001)
  • QIAvac 96 (QIAGEN, cat. no. 19504)

Basic Protocol 3: Enzymatic Removal of Priming Sites

  Materials
  • UltraPure DNase/RNase‐free distilled water (Invitrogen, cat. no. 10977‐023)
  • BtsI (New England Biolabs, cat. no. R0614L) containing:
    • NEBuffer 4
    • BSA
  • 96‐well plate containing the assembly subpools (see protocol 2)
  • MinElute 96 UF PCR purification kit (QIAGEN, cat. no. 28051)
  • EB buffer (10 mM Tris⋅Cl, pH 8.5)
  • Seal‐Rite 1.5‐ml microcentrifuge tubes (USA Scientific, cat. no. 1615‐5500)
  • Vortex Mixer (VWR International, cat. no. 58816‐121)
  • Galaxy mini centrifuge with 1.5/2.0 ml and 0.2 ml tube adapters (VWR, cat. no. 37000‐700)
  • QIAvac 96 (QIAGEN, cat. no. 19504)
  • 96‐reaction thermal cycler (BioRad, cat. no. 186‐1096)

Basic Protocol 4: Assembly of Processed Subpools into Full‐Length Constructs

  Materials
  • Construction primers
  • Assembly subpool‐specific primers ( protocol 1)
  • Ice
  • UltraPure DNase/RNase‐free distilled water (Invitrogen, cat. no. 10977‐023)
  • Phusion high‐fidelity DNA polymerase (New England Biolabs, cat. no. M0530L) containing:
    • HF reaction buffer
  • dNTP solution mix (Enzymatics, cat. no. N205L)
  • QIAquick 96 PCR purification kit (QIAGEN, cat. no. 28183)
  • QIAvac 96 (QIAGEN, cat. no. 19504)
  • Seal‐Rite 1.5‐ml microcentrifuge tubes (USA Scientific, cat. no. 1615‐5500)
  • Vortex mixer (VWR International, cat. no. 58816‐121)
  • Galaxy mini centrifuge with 1.5/2.0 ml and 0.2 ml tube adapters (VWR, cat. no. 37000‐700)
  • Microseal 96‐well skirted low‐profile PCR plates (BioRad, cat. no. MSP‐9601)
  • 96‐Reaction thermal cycler (BioRad, cat. no. 186‐1096)
  • 5‐ml tubes

Support Protocol 1: Gel‐Stab PCR

  Materials
  • Assembly amplification product (see protocol 4)
  • 2% E‐Gel EX gel (Invitrogen, cat. no. G4020‐02)
  • 1‐Kb Plus DNA ladder (Invitrogen, cat. no. 10787‐018)
  • UltraPure DNase/RNase‐free distilled water (Invitrogen, cat. no. 10977‐023)
  • Appropriate construction primers
  • Phusion high‐fidelity DNA polymerase (New England Biolabs, cat. no. M0530L) containing:
    • HF reaction buffer
  • dNTP solution mix (Enzymatics, cat. no. N205L)
  • QIAquick PCR purification kit (QIAGEN, cat. no. 28104)
  • E‐Gel iBase power system (Invitrogen, cat. no. G6400)
  • Thin metal edge or E‐Gel opener (Invitrogen, cat. no. G5300‐01)
  • Safe Imager 2.0 blue light transilluminator (Invitrogen, cat. no. G6600)
  • Seal‐Rite 1.5‐ml microcentrifuge tubes (USA Scientific, cat. no. 1615‐5500)
  • 10‐ or 20‐µl pipet tips
  • 0.2‐ml PCR tubes with flat caps (BioRad, cat. no. TFI‐0201)
  • Galaxy mini centrifuge with 1.5/2.0‐ml and 0.2‐ml tube adapters (VWR, cat. no. 37000‐700)
  • Vortex mixer (VWR International, cat. no. 58816‐121)
  • 96‐Reaction thermal cycler (BioRad, cat. no. 186‐1096)
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Figures

Videos

Literature Cited

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