- Using an enzyme called terminal
deoxynucleotidyl transferase or TdT in a novel
way, researchers at the Berkley lab have found a new way to synthesize DNA
current method is faster, cheaper and better
up DNA synthesis is beneficial for companies trying to sustainably bio-manufacture
useful products, new pharmaceuticals, or tools for more environmentally
A new way to synthesize DNA
sequences through creative
use of enzymes that promises to be faster, cheaper, and more accurate
has been discovered by the
research team at the Berkeley Lab.
discovery was led by graduate students Sebastian Palluk and Daniel Arlow from
the Department of Energy's Joint BioEnergy Institute (JBEI), based at Lawrence
Berkeley National Laboratory (Berkeley Lab) and was published in Nature
in a paper titled "De novo DNA Synthesis Using
‘Production of custom DNA sequences is a fundamental tool for scientific discovery. The current process of DNA synthesis can be slow and unreliable. However, a way to speed up DNA synthesis has been discovered.’
synthesis is at the core of everything we try to do when we build
biology," said JBEI CEO Jay Keasling, the corresponding author on the
paper and also a Berkeley Lab senior faculty scientist. "Sebastian and Dan
have created what I think will be the best way to synthesize DNA since [Marvin]
Caruthers invented solid-phase DNA synthesis almost 40 years ago. What this
means for science is that we can engineer biology much less expensively - and
in new ways - than we would have been able to do in the past."
Let's Get to Know our DNA
deoxyribonucleic acid, is the hereditary material in humans; most of the DNA is
located in the nucleus of the cell and nearly every cell in a person's body has
the same DNA.
The DNA has four chemical bases: adenine (A),
guanine (G), cytosine (C), and thymine (T)
which pair up with each other; A
pairs up with T and C pairs up with G. A DNA
sequence is made up of the combination of the four bases whose order determines
the information available for building and maintaining an organism.
Genes are made up of DNA and act as instructions to
make molecules called proteins. Genes can vary in size from a few hundred DNA
bases to more than 2 million bases. Nowadays, researchers regularly work
with genes of several thousand bases in length.
How are Genes Obtained for Research
Genes can either be
isolated from an existing organism, or bought from a company.
Buying them from a company takes up
time and money. A DNA sequence order from a website can take two weeks to get
delivered while some may not reach on time at all. Plus the purchase at $300
per gene is expensive, if the researcher wants to test a thousand genes.
The students involved in the current
research realized that they were spending many long, tedious hours making DNA
sequences for their experiments, instead of doing the actual experiment.
Solution - Using Enzymes
There is an enzyme called TdT (terminal deoxynucleotidyl transferase)
found in the immune system of vertebrates. It is one of the few enzymes in
nature that can not only write new DNA
from scratch, but is also fast, being able to add 200 bases per minute.
If TdT has to be used to synthesize
the desired sequence, the key requirement is to make it add just one nucleotide (a DNA building block made
up of the bases, sugar and a phosphate group)
, and not any more of the same
nucleotide. That is, it has to stop before it keeps adding the same nucleotide
repeatedly. So far, the nucleotides were modified with special blocking groups
to prevent multiple additions.
However, the problem was that the site
of the enzyme exposed to TdT was not large enough to accept the nucleotide with
a blocking group attached. Attempts to modify the structure of the enzyme to
accommodate the complex have also failed because the technique was compromising
with the activity of the enzyme.
Palluk and Arlow sought a different
method. They tethered one nucleotide to
each TdT enzyme via a cleavable linker
. That way, after extending a DNA
molecule using its tethered nucleotide, the enzyme automatically stops as it
has no other nucleotides available to add. Thus, after the nucleotide is added to the DNA molecule, the enzyme is
. The cycle can begin again with the next nucleotide tethered to
another TdT enzyme.
The approach is clever and
counterintuitive. "Rather than reusing an enzyme as a catalyst, they said,
'Hey, we can make enzymes really inexpensively. Let's just throw it away.' So
the enzyme becomes a reagent rather than a catalyst," he said. "That
kind of thinking then allowed them to do something very different from what's
been proposed in the literature and - I think - accomplish something really
The students were sceptic when they
first demonstrated their method by manually making a DNA sequence of 10 bases.
The method is yet to be optimized, but the students are reasonably confident
that they will be able to eventually
make a gene with 1,000 bases in the first attempt, at many times the speed of
the chemical method
The conventional method now typically
achieves a yield of about 99.5 percent per step. The current proof-of-concept
synthesis with a yield of 98 percent per
, is not quite on par yet, but offers a promising starting point.
The lab dreams to make a gene
- Sebastian Palluk, Daniel H Arlow, Tristan de Rond, Sebastian Barthel, Justine S Kang, Rathin Bector, Hratch M Baghdassarian, Alisa N Truong, Peter W Kim, Anup K Singh, Nathan J Hillson, Jay D Keasling. De novo DNA synthesis using polymerase-nucleotide conjugates. Nature Biotechnology, 2018; DOI: 10.1038/Nbt.417
- What is DNA? - (https://ghr.nlm.nih.gov/primer/basics/dna)