CH391L/S13/DnaAssembly

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Currently, synthetic DNA oligonucleotides less than 60 Bases can be obtained for about #0.35 USD/Base [http://www.idtdna.com/pages/products/dna-rna/custom-dna-oligos]
Currently, synthetic DNA oligonucleotides less than 60 Bases can be obtained for about #0.35 USD/Base [http://www.idtdna.com/pages/products/dna-rna/custom-dna-oligos]
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Revision as of 23:31, 22 January 2013


Contents

Introduction

A large number of parts have been made by the synthetic biology community. Many can be found as part of the Registry of Standard Biological Parts. These modular genetic components are designed to be easy to acquire and assemble to facilitate the building of more complex biological devices. To learn more about the Registry and the biological parts known as BioBricks™, see the entry for the iGEM Registry.

The Registry of Standard Biological Parts is an attempt to create an annotated and characterized repository of biological parts. It is motivated in part because synthetic biologists rely on the ability to make testable biological units. This means that the ability to gather, manipulate, or even create genetic material is vital for "doing" synthetic biology. This page details how to create DNA from small (<60 nts) oligonucleotides to larger genes (~400 nts) to genome sized (~500 ,000 nts) biological units.

Gene Synthesis

Gene synthesis, or artificial gene synthesis, refers to the process of creating a nucleic acid template for a gene in vitro, without the requirement of a preexisting DNA template. Soon after the elucidation of the genetic code and the description of the central dogma of molecular biology, there arose a need to synthesize genes de novo in order to study their biological function both in the test tube and in model organisms. Chemical synthesis of DNA has grown from an expensive and time-consuming process into a viable commercial industry capable of high-throughput manufacture of almost any scale of custom DNA molecules in almost any context. This allows species-specific gene optimization, creation of genes from rare or dangerous sources, and combinatorial assembly of any DNA sequence that can be chemically synthesized, even including non-traditional bases. The most advanced applications of gene synthesis have been applied to the recent creation of completely synthetic minimal genomes in prokaryotes.

Despite nearly four decades of progress in gene synthesis technologies, most DNA sequences used in modern molecular biology are assembled in part or in whole from naturally occurring templates. However this limits the scope and applications to previously existing genes and the results of large-scale genomic surveys of novel genes from nature. Modern gene synthesis relies heavily on advancements in chemical DNA oligonucleotide synthesis, with the primary challenges being scale, cost, fidelity and the eventual assembly of complete gene products.

A directory of commercial gene synthesis providers can be found at Genespace. The company biomatik is not included on this list.

History of Gene Synthesis

Gene synthesis predates the invention of restriction enzymes and molecular cloning techniques by several years. The first gene to be completely synthesized in vitro was a 77-nt alanine transfer RNA by the laboratory of Har Gobind Khorana in 1972 [1]. This was the result of nearly five years of work and resulted in a DNA template without promoter or transcriptional control sequences. The first peptide- and protein-producing synthetic genes were created in 1977 and 1979, respectively [2, 3]. Steady advancement has led to recent synthesis of complete gene clusters tens of thousands of nucleotides in length, and ultimately a bacterial genome approximately 1.2 million bases in length.

Oligonucleotide Synthesis

Regardless of the length of the eventual product, synthetic DNA constructs are built from some combination of short DNA oligonucleotides. These oligonucleotides are later assembled into a complementary DNA duplex, amplified and inserted into their final genetic context.

Oligonucleotides are chemically synthesized from DNA phosphoramidite monomers. Briefly, activated phosphoramidite monomers are added in the 3' to 5' direction using a cyclical activation and blocking chemistry to obtain a DNA polymer linked by phosphodiester bonds.

Image:CH391L_S12_Phosphoramidite.png

Chemical synthesis is currently limited to oligonucleotides of about 200 nt in length.

Cost Analysis

Currently, synthetic DNA oligonucleotides less than 60 Bases can be obtained for about #0.35 USD/Base [1]

carlson_longest_sDNA_2010-thumb-500x457.png

carlson_cost%20per_base_june_2011.png

cost_per_genome.jpg

References
Cost
Time

Molecular Cloning

Restriction Enzyme

BioBricks
BglBricks
list of BioBrick Foundation Standards

Polymerase Chain Reaction

TOPO TA cloning (invitrogen)
SOE (splice by overlap extension) pcr
PCA

Ligation

Recombination/Homology

j5 from Nathan Hillson

-In-Fusion (Clontech) poxvirus DNA polymerase with 3′–5′ exonuclease activity
-In-Fusion BioBrick Assembly
-cold fusion (SBI)

-golden gate
-MoClo [4]
-GoldenBraid

-SLIC sequence and ligation independent cloning T4 DNA polymerase (exonuclease)
-Gibson T5 exonuclease, Phusion polymerase, Taq ligase
-CPEC circular polymerase extension cloning

-SLiCE (Seamless Ligation Cloning Extract) in vitro homologous recombination

Uncategorized
-Fast Seamless Cloning (Dogene)
-CloneEZ kit (Genescript)
-GENEART
-lic
-gateway
-lambda red

Transformation

  • e coli
  • s. cerevisiae

Bacterial Mating

MAGIC

More cloning strategies found here

References

  1. Weber E, Engler C, Gruetzner R, Werner S, and Marillonnet S. . pmid:21364738. PubMed HubMed [Weber2011]
    MoClo

  2. Hughes RA, Miklos AE, and Ellington AD. . pmid:21601682. PubMed HubMed [Hughes2011]
    Gene Synthesis Review

  3. Werner S, Engler C, Weber E, Gruetzner R, and Marillonnet S. . pmid:22126803. PubMed HubMed [Werner2012]
    MoClo

  4. Sarrion-Perdigones A, Falconi EE, Zandalinas SI, Juárez P, Fernández-del-Carmen A, Granell A, and Orzaez D. . pmid:21750718. PubMed HubMed [SarrionPerdigones2011]
    GoldenBraid

  5. Engler C, Kandzia R, and Marillonnet S. . pmid:18985154. PubMed HubMed [Engler2008]
    GoldenGate

  6. Quan J and Tian J. . pmid:19649325. PubMed HubMed [Quan2009]
    CPEC

  7. Gibson DG, Benders GA, Andrews-Pfannkoch C, Denisova EA, Baden-Tillson H, Zaveri J, Stockwell TB, Brownley A, Thomas DW, Algire MA, Merryman C, Young L, Noskov VN, Glass JI, Venter JC, Hutchison CA 3rd, and Smith HO. . pmid:18218864. PubMed HubMed [Gibson2008]
  8. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, and Smith HO. . pmid:19363495. PubMed HubMed [Gibson2009]
    T5 exonuclease recombination

  9. Li MZ and Elledge SJ. . pmid:17293868. PubMed HubMed [Li2007]
    SLIC

  10. Li MZ and Elledge SJ. . pmid:22328425. PubMed HubMed [Li2012]
    SLIC

  11. Sleight SC, Bartley BA, Lieviant JA, and Sauro HM. . pmid:20385581. PubMed HubMed [Sleight2010]
    In-Fusion biobrick

  12. Zhu B, Cai G, Hall EO, and Freeman GJ. . pmid:17907578. PubMed HubMed [Zhu2007]
    in-fusion

  13. Benoit RM, Wilhelm RN, Scherer-Becker D, and Ostermeier C. . pmid:16289702. PubMed HubMed [Benoit2006]
    in-fusion

  14. Geu-Flores F, Nour-Eldin HH, Nielsen MT, and Halkier BA. . pmid:17389646. PubMed HubMed [GeuFlores2007]
    USER

  15. Gibson DG. . pmid:19745056. PubMed HubMed [Gibson2009-2]
    oligonucleotide assembly

  16. Horton RM, Cai ZL, Ho SN, and Pease LR. . pmid:2357375. PubMed HubMed [Horton2009]
    SOEing

  17. Czar MJ, Anderson JC, Bader JS, and Peccoud J. . pmid:19111926. PubMed HubMed [Czar2009]
    review

  18. Stemmer WP, Crameri A, Ha KD, Brennan TM, and Heyneker HL. . pmid:7590320. PubMed HubMed [Stemmer1995]
    PCA

  19. Aslanidis C and de Jong PJ. . pmid:2235490. PubMed HubMed [Aslanidis1990]
    LIC

  20. Aslanidis C, de Jong PJ, and Schmitz G. . pmid:7580902. PubMed HubMed [Aslanidis1994]
    LIC

  21. Li C and Evans RM. . pmid:9321675. PubMed HubMed [Li1997]
    LIC

  22. Angrand PO, Daigle N, van der Hoeven F, Schöler HR, and Stewart AF. . pmid:10446259. PubMed HubMed [Angrand1999]
    lambda Red recombinase

  23. Hartley JL, Temple GF, and Brasch MA. . pmid:11076863. PubMed HubMed [Hartley2000]
    Gateway lambda Int

  24. Khalil AM, Julius JA, and Bachant J. . pmid:17702758. PubMed HubMed [Khalil2007]
    Gateway lambda Cre

  25. Larionov V, Kouprina N, Graves J, Chen XN, Korenberg JR, and Resnick MA. . pmid:8552668. PubMed HubMed [Larionov1996]
    Transformation-associated recombination (TAR) cloning

  26. j5 DNA Assembly Design Automation Software doi: 10.1021/sb2000116 [Hillson2012]
  27. Gibson DG, Glass JI, Lartigue C, Noskov VN, Chuang RY, Algire MA, Benders GA, Montague MG, Ma L, Moodie MM, Merryman C, Vashee S, Krishnakumar R, Assad-Garcia N, Andrews-Pfannkoch C, Denisova EA, Young L, Qi ZQ, Segall-Shapiro TH, Calvey CH, Parmar PP, Hutchison CA 3rd, Smith HO, and Venter JC. . pmid:20488990. PubMed HubMed [Gibson2010]
    genome replacement

  28. Li MZ and Elledge SJ. . pmid:15731760. PubMed HubMed [Li2005]
    MAGIC, bacterial mating approach

  29. Zhang Y, Werling U, and Edelmann W. . pmid:22241772. PubMed HubMed [Zhang2012]
    SLiCe

All Medline abstracts: PubMed HubMed
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