Improved Technique Produces Array of Human-made DNA Sequences
January 26, 2011 12:43 PM
(Source: Duke University Photography )
New faster technique could lead to the development of new types of drugs
have created an improved technique that is capable of producing nearly an unlimited array of human-made DNA sequences quickly and easily.
Ashutosh Chilkoti, study leader and Theo Pilkington Professor of Biomedical Engineering at Duke University's Pratt School of Engineering, along with Daniel Callahan, of Duke University, and graduate students Miriam Amiram and García Quiroz, have developed a method for creating human-made DNA sequences faster in order to produce repetitive proteins, which are used to make new bioengineered tissues and drugs.
Traditional techniques for creating DNA sequences are not robust or very slow, which has prevented the development of new classes of protein-based polymers.
But now, the Duke University researchers have created
a new process
called overlap-extension rolling circle amplification. The process attaches a macromolecule, or polymer, to a protein, which allows the protein to maintain activity in the bloodstream longer and increases effectiveness. But instead of building these protein building blocks individually the way traditional methods do now, this new technique can make almost an unlimited number of them.
"Depending on how complicated you want the polymer sequence to be, there are an infinite number of combinations you could make," said Chilkoti. "We haven't even begun to look at all the sequences that can be made or the unique properties they might have."
One beneficial trait to this new technique is that it is an improvement to already-existing methods, which makes it inexpensive to use in the laboratory.
"A very popular method for making tandem copies of
involves inserting them iteratively into a bacterial plasmid," said Amiram. "After the vector has grown in size, the copies of the sequence are cut out using enzymes and the process is repeated to generate a larger polymer. It is a very time-consuming process.
"With this new method, you don't get just one product, but many. This should help us to make large libraries of proteins, which we can use to rapidly screen new combinations. This powerful strategy generates
libraries of repetitive genes
over a wide range of molecular weights in a 'one-pot' parallel format."
To test this improved technique, the Duke University researchers synthesized genes located in two different classes of protein-polymers. The first was protein-polymer combinations for elastin, which is found in connective tissue. The second synthesized genes in glucagon-like peptide-1 analogs "to show variable pharmokinetic properties. Researchers were successfully able to synthesize genes in both cases.
"This could help remove one of the biggest stumbling blocks we face in producing these drugs," said Chilkoti. "You can't make the proteins without genes, which act as the software directing the protein's production. Instead of building each sequence individually, as is done now, we can literally make hundreds, each with subtle differences."
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