In Vitro Seamless Stack Enzymatic Assembly of DNA Molecules Based on a Strategy Involving Splicing of Restriction Sites

Scientific Reports
Dong YuDingyang Yuan

Abstract

The standard binary enzymatic assembly, which operates by inserting one DNA fragment into a plasmid, has a higher assembly success rate than the polynary enzymatic assembly, which inserts two or more fragments into the plasmid. However, it often leaves a nucleotide scar at the junction site. When a large DNA molecule is assembled stepwise into a backbone plasmid in a random piecewise manner, the scars will damage the structure of the original DNA sequence in the final assembled plasmids. Here, we propose an in vitro Seamless Stack Enzymatic Assembly (SSEA) method, a novel binary enzymatic assembly method involving a seamless strategy of splicing restriction sites via a stepwise process of multiple enzymatic reactions that does not leave nucleotide scars at the junction sites. We have demonstrated the success and versatility of this method through the assembly of 1) a 4.98 kb DNA molecule in the 5' → 3' direction using BamHI to generate the sticky end of the assembly entrance, 2) a 7.09 kb DNA molecule in the 3' → 5' direction using SmaI to generate the blunt end of the assembly entrance, and 3) an 11.88 kb DNA molecule by changing the assembly entrance.

References

Oct 25, 1990·Nucleic Acids Research·C Aslanidis, P J de Jong
Nov 15, 2000·Genome Research·J L HartleyM A Brasch
Feb 13, 2007·Nature Methods·Mamie Z Li, Stephen J Elledge
Nov 6, 2008·PloS One·Carola EnglerSylvestre Marillonnet
Apr 14, 2009·Nature Methods·Daniel G GibsonHamilton O Smith
Mar 3, 2010·Nucleic Acids Research·William J BlakeBrian M Baynes
Apr 14, 2010·Nucleic Acids Research·Sean C SleightHerbert M Sauro
Jan 20, 2011·Integrative Biology : Quantitative Biosciences From Nano to Macro·Tom EllisGeoff S Baldwin
Mar 3, 2011·PloS One·Ernst WeberSylvestre Marillonnet
Mar 17, 2012·Nucleic Acids Research·Jonathan Leo Schmid-BurgkYaakov Benenson
May 29, 2012·Current Opinion in Chemical Biology·Siying MaJingdong Tian
Jun 12, 2012·PloS One·Arjen J Jakobi, Eric G Huizinga
May 10, 2015·Nucleic Acids Research·Leslie A MitchellJef D Boeke
Sep 12, 2015·PloS One·Hannes M BeyerMatias D Zurbriggen

❮ Previous
Next ❯

Citations

Feb 28, 2020·Scientific Reports·Dong YuDingyang Yuan

❮ Previous
Next ❯

Methods Mentioned

BETA
PCR
electrophoresis

Software Mentioned

DNAssist
DNAssit
SSEA

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.