One-Pot Golden Gate Cloning via Methylation and Chemically Modified Assisted Tailorable Ends (McMaster)

APPLICATIONS OF TECHNOLOGY:

DNA Assembly & Cloning
Biological Research
Sequencing
Protein Expression

ADVANTAGES:

One pot cloning, regardless of internal restriction sites
Assembly using various restriction enzymes

BACKGROUND:

Golden Gate assembly is the preferred DNA assembly technique for repetitive DNA elements and secondary structure, as well as for hierarchical assembly. In order to assemble DNA parts with internal restriction sites, assembly techniques independent of using restriction enzymes or more complex techniques are needed. Golden Gate cloning, for example, requires the presence of type IIS enzyme restriction sites at the ends of the fragments and vector. Further, the DNA fragments and recipient vector must lack those same restriction sites in internal sequences. There is a need for a more streamlined DNA assembly process for fragments containing internal restriction sites.

TECHNOLOGY OVERVIEW:

This invention includes several restriction enzyme techniques for DNA assembly of fragments containing internal restriction sites. The first technique is a modification of the Golden Gate method in which the assembly parts being run through PCR have modified nucleotides (dNTPs) to silence internal sites while leaving the unmodified or hemi-modified terminal recognition sites cleavable.

The second technique includes methyltransferase-driven modification of DNA assembly parts without PCR to silence the internal recognition sites, ligation of unmodified adapters containing non-silenced sites, and subsequent assembly from these non-silenced sites. The first and second techniques can be hybridized to tailor to assembly needs.

DEVELOPMENT STAGE:

Proof of concept

INVENTORS: