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In situ overlap and sequence synthesis during DNA assembly
- Source :
- ACS Synthetic Biology
- Publication Year :
- 2013
-
Abstract
- Modern cloning methods are independent from restriction enzyme recognition sites. However, nearly all current cloning methods still require the introduction of overlaps by PCR, which can introduce undesired mutations. Here, we investigated whether overlaps needed for DNA assembly can be synthesized in situ and we tested if the de novo synthesis of sequences can be simultaneously combined with the assembly of larger double-stranded DNA fragments. We showed in a set of 44 cloning experiments that overlaps of 20 bp needed for DNA assembly can be synthesized in situ from single-stranded oligonucleotides. Short sequences of 30–255 bp can be synthesized from single-stranded oligonucleotides concurrently with DNA assembly, and both techniques can be combined. The assembly of similar constructs by state-of-the-art techniques would have required multiple rounds of cloning or tedious sample preparations, whereas our approach is a one-step reaction.
- Subjects :
- Genetic Vectors
Biomedical Engineering
Cloning vector
cloning
Computational biology
Molecular cloning
Biology
de novo synthesis
Polymerase cycling assembly
Biochemistry, Genetics and Molecular Biology (miscellaneous)
overlaps
03 medical and health sciences
chemistry.chemical_compound
Multiple cloning site
Escherichia coli
Technical Note
Cloning, Molecular
030304 developmental biology
Genetics
0303 health sciences
oligonucleotides
Oligonucleotide
030302 biochemistry & molecular biology
fungi
food and beverages
General Medicine
DNA
in situ DNA assembly
Restriction enzyme
chemistry
In vitro recombination
Subjects
Details
- ISSN :
- 21615063
- Volume :
- 2
- Issue :
- 12
- Database :
- OpenAIRE
- Journal :
- ACS synthetic biology
- Accession number :
- edsair.doi.dedup.....cf2dd7e4c688976279a2d3cd7c13b6be