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More cloning strategies found [http://dnasu.asu.edu/DNASU/CloningStrategies.jsp here]
More cloning strategies found [http://dnasu.asu.edu/DNASU/CloningStrategies.jsp here]
===Links of Interest===
===Links of Interest===
Revision as of 05:29, 28 January 2013
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.
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, 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 . 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 .
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 enzymes recognize a specific nucleotide sequence and then cut the DNA in such a way that results in a double stranded break. If the enzyme cuts within the recognition site, it is classified as a Type I restriction enzyme. If the enzyme cuts outside of the restriction site, the enzyme is classified as a Type II restriction enzyme. Restriction enzymes can also be classified by if the DNA break results in a straight cut resulting in a blunt end or with a jagged cut resulting in a sticky end.
For example: EcoRI digestion produces "sticky" ends,
whereas SmaI restriction enzyme cleavage produces "blunt" ends:
The Registry of Biological Parts has developed [The_BioBricks_Foundation:Standards/Technical/Formats | standards] for the type and position of restriction enzyme sites to be used when building DNA using restriction enzymes in order to ensure compatibility in the connecting of biological parts. Two of the more popular standards are named and listed below:
One modification to the standard restriction enzyme method is the use of an enzyme that recognizes a non-palindromic sequence. This results in the ability to cut with a single restriction enzyme and still maintain directionality of the biological part.
- CpoI directional cloning
Other methods have been developed that utilize Type II restriction enzymes. These enzymes cut away from their recognition site. This strategy has the advantage over Type I enzymes in their reduction of a "scar" sequence and ability to generate combinatorial libraries.
- golden gate
- MoClo 
Polymerase Chain Reaction
Polymerase Chain Reaction (PCR) is a fast and reliable way of making multiple copies of specific sequences of DNA. The PCR methods typically allows for relatively simple and fast construction of genes by linking multiple shorter oligonucleotides together in a series of steps where the earlier PCR product acts as a primer that results in a longer DNA fragment that itself can act as a primer for subsequent rounds of PCR. An exception is TOPO TA cloning, which utilizes the propert of Taq polymerase to leave a single A nucleotide overhang after the PCR reaction. In combination with an optimized vector with a T nucleotide overhang and a topoisomerase that recognizes the vector at the T overhang position, the insert and vector that are annealed at the T and A nucleotides are sealed into a circular vector efficiently.
- Ligation Independent Cloning (LIC) 
- TOPO TA Cloning (Invitrogen) 
- Splice by Overlap Extension (SOE) PCR 
- Polymerase Cycling Assembly (PCA) 
- In-Fusion (Clontech) poxvirus DNA polymerase with 3′–5′ exonuclease activity 
- In-Fusion BioBrick Assembly 
- cold fusion (SBI)
- Cre/Lox P1 phage (Clontech)
- att lambda (gateway)
- CloneEZ kit (Genescript) , 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
-MAGIC (bacterial mating) 
-Recombineering lambda red
More cloning strategies found here
PCR-based gene assembly strategies are similar to ligation-mediated assembly, except that oligonucleotide precursors are linked together leaving gaps that must be filled in by DNA polymerase. In the most common method, polymerase cycling assembly (PCA), DNA oligonucleotides are designed to be part of either the top or bottom of the final DNA duplex. Cycles of annealing and polymerase extension result in a growing DNA duplex built from smaller oligonucleotide fragments. A final round of PCR is done with constant primers that amplify the complete desired gene product. Initial oligo linkage is done for 20-30 cycles and full-length PCR is carried out for 20-30 additional cycles.
Oligos must be designed carefully to have similar melting temperatures and be free of interfering secondary structures (high GC content, hairpins, etc.)
Moving Forward: Scaling Up Gene Synthesis
The methods below are not new methods and some are already being used commercially. What they offer for the future are methods for potentially scaling up production while decreasing costs. As mentioned before, cost and speed are major limitations preventing de novo DNA synthesis from becoming standard practice.
An alternative gene synthesis technique using solid-phase synthesis of a DNA duplex has been created by several companies, including Blue Heron. In these methods, DNA oligonucleotides are synthesized as in other techniques, they are annealed into complementary short duplexes without overhanging ends, and these duplexes are chemically ligated on a solid phase such as a liquid chromatography column. The complete synthetic DNA duplex is then eluted and used for downstream processes, such as cloning or gene expression.
DNA oligonucleotides have become a commodity and the price per base has been steadily dropping. But there is still great expense involved in large-scale gene synthesis. For a several kilobase gene fragment, oligo cost alone can still exceed $1,000. To over come this hurdle, multiplex parallel gene synthesis technologies are under development, including inkjet DNA printers, photolithography and electrochemical parallel synthesis.
Microfluidic microchips are a common multiplexing solution. In the method of Tian et al. , oligonucleotides are synthesized while coupled to a microchip at their 3' end. The overall process is similar to traditional oligo synthesis, except that individual wells are photoactivated before addition of the next base onto the 5' end. After chemical cleavage of the complete oligo from the microchip, they are hybridized and purified using complementary "quality assurance" oligos bound to magnetic beads. This method eliminates oligos that have either incorporated extra bases or undergone deletions. Final yield from each synthesis well is only around 5 fmol of oligo, which is not sufficient for downstream applications. A final amplification step using common PCR primers to all oligos increases yields up to a million-fold. Oligos can then be assembled by overlap PCR-based methods such as polymerase assembly multiplexing, or PAM. This method has resulted in the synthesis, amplification and expression of a 14.5 kb, 21-gene 30S ribosomal protein operon from E. coli.
One major roadblock remaining in synthetic gene synthesis is overcoming natural error rates in DNA synthesis chemistry. Phosphoramidite oligo synthesis currently has an error rate of 1 in ~160. This results in a high enough error rate in assembled genes that screening and sequencing are major cost bottlenecks, as is the repair or re-synthesis of mutated genes. While the development of cheap and high-throughput sequencing technologies will streamline the screening process, oligo synthesis error rates must also be reduced.
Currently, oligo pools are often screened against complementary reference libraries that can be reused. Oligos that do not completely base-pair with their designed sequence are either washed away in purification steps or are cleaved by DNA mismatch repair proteins due to their imperfect base-pairing. This has currently decreased error rates to 1 in 106, on the order of error rates in DNA polymerases.
Links of Interest
- Gibthon - Gibson assembly design program
- j5: A tool for designing DNA assembly with recombination from Nathan Hillson The manual has an excellent overview of recombination-based cloning strategies like SLIC and Gibson. 
- Khorana HG, Agarwal KL, Büchi H, Caruthers MH, Gupta NK, Kleppe K, Kumar A, Otsuka E, RajBhandary UL, Van de Sande JH, Sgaramella V, Terao T, Weber H, and Yamada T. . pmid:4571075.
- Itakura K, Hirose T, Crea R, Riggs AD, Heyneker HL, Bolivar F, and Boyer HW. . pmid:412251.
- Goeddel DV, Kleid DG, Bolivar F, Heyneker HL, Yansura DG, Crea R, Hirose T, Kraszewski A, Itakura K, and Riggs AD. . pmid:85300.
- 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.
- Weber E, Engler C, Gruetzner R, Werner S, and Marillonnet S. . pmid:21364738.
- Aslanidis C and de Jong PJ. . pmid:2235490.
- Holton TA and Graham MW. . pmid:2020554.
- Shuman S. . pmid:1658796.
- Higuchi R, Krummel B, and Saiki RK. . pmid:3045756.
- Stemmer WP, Crameri A, Ha KD, Brennan TM, and Heyneker HL. . pmid:7590320.
- Zhu B, Cai G, Hall EO, and Freeman GJ. . pmid:17907578.
- Benoit RM, Wilhelm RN, Scherer-Becker D, and Ostermeier C. . pmid:16289702.
- Sleight SC, Bartley BA, Lieviant JA, and Sauro HM. . pmid:20385581.
- Li MZ and Elledge SJ. . pmid:15731760.
MAGIC, bacterial mating approach
- Tian J, Gong H, Sheng N, Zhou X, Gulari E, Gao X, and Church G. . pmid:15616567.
- j5 DNA Assembly Design Automation Software doi: 10.1021/sb2000116
- 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.
- Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CA 3rd, and Smith HO. . pmid:19363495.
oligonucleotide assembly in vitro
- Gibson DG. . pmid:19745056.
oligonucleotide assembly in yeast
- Gibson DG, Smith HO, Hutchison CA 3rd, Venter JC, and Merryman C. . pmid:20935651.
- Gibson DG. . pmid:21601685.
- Dymond JS, Richardson SM, Coombes CE, Babatz T, Muller H, Annaluru N, Blake WJ, Schwerzmann JW, Dai J, Lindstrom DL, Boeke AC, Gottschling DE, Chandrasegaran S, Bader JS, and Boeke JD. . pmid:21918511.
- Hughes RA, Miklos AE, and Ellington AD. . pmid:21601682.
Gene Synthesis Review
- Werner S, Engler C, Weber E, Gruetzner R, and Marillonnet S. . pmid:22126803.
- Sarrion-Perdigones A, Falconi EE, Zandalinas SI, Juárez P, Fernández-del-Carmen A, Granell A, and Orzaez D. . pmid:21750718.
- Engler C, Kandzia R, and Marillonnet S. . pmid:18985154.
- Quan J and Tian J. . pmid:19649325.
//T5 exonuclease recombination
- Li MZ and Elledge SJ. . pmid:17293868.
- Li MZ and Elledge SJ. . pmid:22328425.
- Geu-Flores F, Nour-Eldin HH, Nielsen MT, and Halkier BA. . pmid:17389646.
- Horton RM, Cai ZL, Ho SN, and Pease LR. . pmid:2357375.
- Czar MJ, Anderson JC, Bader JS, and Peccoud J. . pmid:19111926.
- Aslanidis C, de Jong PJ, and Schmitz G. . pmid:7580902.
- Li C and Evans RM. . pmid:9321675.
- Angrand PO, Daigle N, van der Hoeven F, Schöler HR, and Stewart AF. . pmid:10446259.
lambda Red recombinase
- Hartley JL, Temple GF, and Brasch MA. . pmid:11076863.
Gateway lambda Int
- Khalil AM, Julius JA, and Bachant J. . pmid:17702758.
Gateway lambda Cre
- Larionov V, Kouprina N, Graves J, Chen XN, Korenberg JR, and Resnick MA. . pmid:8552668.
Transformation-associated recombination (TAR) cloning
- Zhang Y, Werling U, and Edelmann W. . pmid:22241772.