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DNA Synthesis Made Faster and Cheaper
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DNA Synthesis Made Faster and Cheaper

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Highlights:
  • 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
  • The current method is faster, cheaper and better
  • Speeding up DNA synthesis is beneficial for companies trying to sustainably bio-manufacture useful products, new pharmaceuticals, or tools for more environmentally friendly agriculture

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.

The 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 Biotechnology in a paper titled "De novo DNA Synthesis Using Polymerase-Nucleotide Conjugates."

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"DNA 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


DNA, or 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.
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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 Creatively


There is an enzyme called TdT (terminal deoxynucleotidyl transferase) which is 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 cleaved off. 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 important."

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 step, is not quite on par yet, but offers a promising starting point.

The lab dreams to make a gene overnight.

References :
  1. 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
  2. What is DNA? - (https:ghr.nlm.nih.gov/primer/basics/dna)


Source: Medindia

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