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DNA synthesis and molecular cloning are tools used by synthetic biologists to create the biological "parts" needed to design and engineer biological devices and systems.



As described before, synthetic biology captures a diverse, multi-disciplinary field. No matter which definition(s) becomes accepted, the ability to make and manipulate DNA is a vital component to practicing synthetic biology.

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. While the parts registry is a useful resource, it is not comprehensive. The ability to manipulate and create genetic material is a necessary skill for being a successful synthetic biologists. 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. Many of the methods found here are the basis for the construction of the registry itself.

DNA Synthesis

Oligonucleotide Synthesis

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.


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

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.

An extensive, but not comprehensive, directory of commercial gene synthesis providers can be found at Genespace.


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 1.1 million bases in length [4].

Longest Published Synthetic DNA [1]
Longest Published Synthetic DNA [1]

Molecular Cloning

It is impractical for most synthetic biologists to synthesize more than several kilobases of completely synthetic DNA. It is often desirable to build bigger pieces at lower costs and faster speeds than de novo synthesis is currently able to accomplish.

Over the years, many different strategies have been developed to assemble DNA in flexible ways that suit different purposes. These strategies typically employ purification of enzymes that are known to modify DNA in specific ways and these methods of action can be exploited for designing and building specific sequences of DNA. For example, restriction enzymes are used to cut DNA in a specific manner upon recognition of a specific nucleotide sequence. Polymerases and endonucleases add or remove nucleotides to make double stranded DNA from a single stranded template or to create single stranded DNA from double stranded DNA. Many of the modern techniques take advantage of recombination machinery that break DNA from one location to reattach it to another location. In other cases, the endogenous enzymes in a host are utilized to manipulate DNA without the need for prior purification. For example, in some methods endogenous DNA ligase is used to repair single stranded breaks (known as "nicks") to complete the formation of fully circular DNA.

Ultimately, these methods generally require transformation into a host where endogenous enzymes are used to complete the genetic manipulation and replicate (clone) the genetic material. This allows for the expression of the desired proteins to test the ability of the engineered system or for the purification of the genetic material itself such that it can be used for further manipulation, study, or storage.

While there are many specific protocols for the numerous methods of cloning, most share reasonable overlap in their underlying mechanisms of action. Broadly speaking, methods may rely primarily on restriction enzymes, polymerase chain reaction (PCR), or on homologous recombination.

Restriction Enzyme

Restriction enzymes are

  • BioBricks
  • BglBricks

list of BioBrick Foundation Standards

  • CpoI directional cloning
  • golden gate
  • MoClo [5]
  • GoldenBraid

Polymerase Chain Reaction

Ligation Independent Cloning (LIC) [6]
TOPO TA Cloning (Invitrogen) [7][8]
Splice by Overlap Extension (SOE) PCR [9]
Polymerase Cycling Assembly (PCA) [10]


  • In-Fusion (Clontech) poxvirus DNA polymerase with 3′–5′ exonuclease activity [11][12]
  • In-Fusion BioBrick Assembly [13]
  • cold fusion (SBI)
  • Cre/Lox P1 phage (Clontech)
  • att lambda (gateway)
  • CloneEZ kit (Genescript) [2], recombination around a linearized vector
  • GENEART Seamless Cloning (Life Technologies previously Invitrogen previously DoGene)
  • 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

In Vivo

-MAGIC (bacterial mating) [14]
-Recombineering lambda red

More cloning strategies found here

Links of Interest


Error fetching PMID 4571075:
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  1. Error fetching PMID 4571075: [Khorana1972]
  2. Error fetching PMID 412251: [Itakura1977]
  3. Error fetching PMID 85300: [Goeddell1979]
  4. Error fetching PMID 20488990: [Gibson2010]
    genome replacement

  5. Error fetching PMID 21364738: [Weber2011]

  6. Error fetching PMID 2235490: [Aslanidis1990]

  7. Error fetching PMID 2020554: [Holton1991]
    TA cloning

  8. Error fetching PMID 1658796: [Shuman1991]

  9. Error fetching PMID 3045756: [Higuchi1988]

  10. Error fetching PMID 7590320: [Stemmer1995]

  11. Error fetching PMID 17907578: [Zhu2007]

  12. Error fetching PMID 16289702: [Benoit2006]

  13. Error fetching PMID 20385581: [Sleight2010]
    In-Fusion biobrick

  14. Error fetching PMID 15731760: [Li2005]
    MAGIC, bacterial mating approach

  15. j5 DNA Assembly Design Automation Software doi: 10.1021/sb2000116


  16. Error fetching PMID 15616567: [Tian2004]
  17. Error fetching PMID 18218864: [Gibson2008]
  18. Error fetching PMID 19363495: [Gibson2009]
    oligonucleotide assembly in vitro

  19. Error fetching PMID 19745056: [Gibson2009b]
    oligonucleotide assembly in yeast

  20. Error fetching PMID 20935651: [Gibson2010b]
  21. Error fetching PMID 21601685: [Gibson2011]
    MIE paper

  22. Error fetching PMID 21918511: [Dymond2011]
  23. Error fetching PMID 21601682: [Hughes2011]
    Gene Synthesis Review

  24. Error fetching PMID 22126803: [Werner2012]

  25. Error fetching PMID 21750718: [SarrionPerdigones2011]

  26. Error fetching PMID 18985154: [Engler2008]

  27. Error fetching PMID 19649325: [Quan2009]

    //T5 exonuclease recombination

  28. Error fetching PMID 17293868: [Li2007]

  29. Error fetching PMID 22328425: [Li2012]

  30. Error fetching PMID 17389646: [GeuFlores2007]

  31. Error fetching PMID 2357375: [Horton2009]

  32. Error fetching PMID 19111926: [Czar2009]

  33. Error fetching PMID 7580902: [Aslanidis1994]

  34. Error fetching PMID 9321675: [Li1997]

  35. Error fetching PMID 10446259: [Angrand1999]
    lambda Red recombinase

  36. Error fetching PMID 11076863: [Hartley2000]
    Gateway lambda Int

  37. Error fetching PMID 17702758: [Khalil2007]
    Gateway lambda Cre

  38. Error fetching PMID 8552668: [Larionov1996]
    Transformation-associated recombination (TAR) cloning

  39. Error fetching PMID 22241772: [Zhang2012]

All Medline abstracts: PubMed HubMed
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