Advances in Synthetic DNA
An Overview and Discussion of Recent Innovations

By
Mario Blanco, PhD
Nanopec, Co-Founder & CEO


Introduction


DNA synthesis occurs in all eukaryotes and prokaryotes. The accurate synthesis of DNA is important to avoid mutations in DNA. In humans, mutations could lead to diseases such as cancer. For these and other fundamental reasons, DNA biosynthesis, and the machinery involved in vivo, has been studied extensively since the 1950’s.
 

DNA  biosynthesis in nature occurs via the polymerase chain reaction. In vitro DNA amplification or gene synthesis - physically creating artificial gene sequences, using enzymes in solution, is very slow and prone to errors. Purely chemical means to create DNA are now available that supersede in length and accuracy these biosynthesis methods. Though each type of chemical synthesis is very different, they have several important features in common. Single nucleotides can be joined to form oligonucleotides that can act as a DNA template for gene manufacture. Entire genes, consisting of thousands of nucleotide (nt) bases are now possible. Gene chemical synthesis, however, does not require a DNA template and genes can be assembled de novo.


The need for gene sequence manufacturing will change significantly in the years to come.  In
specialized Academic areas such as creating de novo organisms, CRISPR technology will make signficiant contributions to the field.  CRISPR is a family of DNA sequences found in the genomes of prokaryotic organisms such as bacteria and archaea, will change how genes are altered, edited and manipulated directly into living organisms. Cas9 enzymes together with CRISPR sequences form the basis of a technology known as CRISPR-Cas9 that can be used to edit genes within organisms. This editing process has a wide variety of applications including basic biological research, development of biotechnology products, and treatment of diseases.  However, research in this area as well as in precision medicine, gene sequencing and diagnostics will continue to be strongly dependent on chemical synthesis methods for DNA, oligonucleotide synthesis.


A growing use, and perhaps commercially the most feasible, form of DNA synthesis is the creation of
primers and probes for PCR.  PCR works by using a relatively short (100 nt to 200 nt) single stranded DNA
template. The DNA double helix unwinds during replication, exposing unpaired bases for new nucleotides to hydrogen bond to. Without the primer template PCR is unable to amplify DNA. Probes are also relatively short oligonucleotide sequences that are capped with a fluorophore and a quencher. Their
sequence rarely change in the ends, which generate a hairpin structure to keep the fluorophore from emitting unless the mid-section of the probe finds a complementary strand of DNA to bind. In this case the hairpin is unfolded and the quencher and fluorophore separated by the exonuclease activity of DNA polymerase during amplification. As a result the solution becomes fluorescent which indicates the presence of the sought DNA sequence. These two areas are two of the key roles for oligonucleotide DNA synthesis. One additional area that has become relevant after a recent publication on the efforts to synthesize a commercial COVID-19 vaccine, the emerging field of mRNA vaccines.

In a series of two white papers we summarize the various established methods of oligonucleotide synthesis and take a look at emerging technologies, their advantages and their limitations.

Request a full copy of this white paper to:  mchaudoir@nanopec.com

NANOPEC

9040 S Rita Rd

Suite 1270

Tucson, AZ 85747

USA

+1 520 382 3273

info@nanopec.com

©2020 by NANOPEC