THE HUMAN PROTEIN ATLAS BLOG

Solid-phase cloning for high-throughput assembly

2015-12-08
DNA Expression vector High-throughput cloning


Attachment of DNA fragments onto a growing strand of DNA linked to a paramagnetic bead

Solid-phase cloning for high-throughput assembly of single and multiple DNA parts

Simple and automated workflows for DNA assembly are necessary in large-scale projects such as the Human Protein Atlas. For this reason, we have developed two automated methods for swift and convenient high-throughput assembly of DNA into expression vector constructs.

By using paramagnetic beads as a support for the DNA, we can perform construct assembly on a liquid handler robot and furthermore eliminate the need for prior purification of PCR products by spin or gel purification.

The first method, RE-based SPC, is a highly efficient cloning method utilizing restriction enzymes and has been used to clone over 60 000 human gene fragments within the Human Protein Atlas. The effect of insert sequence characteristics (length, GC content, and complexity) was analyzed and a high GC content was concluded to have the most negative impact on the success rate of the cloning.

For the second method, head-to tail SPC, single stranded DNA is allowed to hybridize through complementary end-regions and are subsequently extended by a proof-reading polymerase. With this approach, multiple DNA fragments can be joined in a sequence independent fashion without having additional bases introduced between the pieces.

Read more about the automated methods for assembly of DNA into expression vector constructs here.