# Key Points from #hplusroadmap Discussion on DNA Assembly and Biotechnology

• Researchers discussed advanced DNA assembly technologies, including in vivo homologous recombination methods that can achieve 95% correct yield when assembling nine fragments with 60-bp overlapping regions.

• E. coli is increasingly being used for homologous recombination instead of yeast, suggesting a shift in preferred organisms for DNA assembly work in synthetic biology.

• The group explored using recombinases for DNA assembly, noting how these enzymes can invade double-stranded DNA and displace one strand, with optimal function using PCR-length primers of 24-30 nucleotides.

• XNA (xeno nucleic acids) and phosphorothioates were discussed as methods to protect DNA from exonuclease degradation, particularly for protecting the last 2-3 bases of primers in assembly reactions.

• An intriguing neurological mechanism was highlighted where some neurotransmitters are regulated by base excision repair in neurons, with calculations suggesting this would damage DNA after approximately 100 years, prompting discussion about replacing this with less destructive mechanisms.

• The conversation compared genome editing technologies (zinc finger nucleases, TALENs, and CRISPR), noting CRISPR's practical advantage of using nucleic acid guides rather than requiring new proteins for each target.

• Participants discussed whole genome sequencing costs for novel organisms, estimating six-figure costs for decent coverage of an Alaskan frog with freeze/thaw capabilities, with suggestions to start with exome sequencing and use related species as assembly templates.