Nav: Home

Copying made easy

March 12, 2019

Whether revealing a perpetrator with DNA evidence, diagnosing a pathogen, classifying a paleontological discovery, or determining paternity, the duplication of nucleic acids (amplification) is indispensable. In the journal Angewandte Chemie, scientists have now introduced a new, very simple, yet highly sensitive and reliable method that avoids the usual heating and cooling steps, as well as complicated instruments. The reagents can be freeze-dried, allowing this universal method to be used outside of the laboratory.

The most commonly used amplification method is the polymerase chain reaction (PCR), which is based on the repetition of multiple thermal cycles in special instruments that have a high demand for power. It is difficult to perform outside of a laboratory, at a patient's bedside or in a remote location, for example. Alternative methods without thermal cycles are often complicated or not sensitive enough, require expensive reagents, or are not broadly applicable.

Researchers working with Bin-Cheng Yin and Bang-Ce Ye at East China University of Science & Technology, Shanghai, China, have now developed a new, inexpensive method: Called the Cas9n-based amplification reaction (Cas9nAR), it consists of a single step in homogeneous solution, and takes place at a constant temperature of 37 °C.

In this approach the researchers use components from the "immune system" of bacteria. When bacteria are infected by a virus, for example, they cut up the foreign genetic material into little bits and introduce them into specific areas of their own genome. In the case of a subsequent infection, the bacterium's RNA strands "recognize" these sequences and direct special "genetic scissors" to cut up the foreign DNA. These tools have also been employed in modern genetic engineering.

Yin, Ye, and their co-workers have altered the genetic scissor known as Cas9 so that it no longer completely cuts through DNA. Instead, it cuts through only one strand, introducing a "nick". This type of enzyme is called a "nickase". Like in the bacterial system, Cas9 nickase binds to an RNA strand, which determines the location of the nick. This RNA can be made so that it recognizes a DNA sequence characteristic of a pathogen, for example. The Cas9 nickase then nicks the immediately adjacent DNA.

For the new technique, the researchers produced two different Cas9 nickase RNA complexes, which nick the DNA in two different places. A polymerase commonly used in PCR (exo(?) Klenow polymerase) complements the cut strand starting at the first nick, setting the old strand free, piece by piece, until it reaches the second nick. The newly completed DNA is repeatedly nicked and complemented by the nickase complex. The short single strands this process releases become the starting point for further amplification in a second cycle. In addition to the nickase complex and the polymerase, the only things required are two suitable primers as starting points for the copies.

Tests with a fragment of bacterial genomic DNA demonstrated that the target sequence was precisely recognized and amplified. In a volume of 20 μl, it was possible to detect a single molecule. Differences of a single nucleotide within a gene could be detected with high specificity.
-end-
About the Author

Dr Bang-Ce Ye is a professor at the East China University of Science and Technology in Shanghai. His research focuses on analytical biotechnology and engineering biology, being devoted to the development of innovative methods or tools to apply in clinical diagnosis, environmental monitoring, synthetic biology, etc.

mailto:binchengyin@ecust.edu.cn

Wiley

Related Dna Articles:

Penn State DNA ladders: Inexpensive molecular rulers for DNA research
New license-free tools will allow researchers to estimate the size of DNA fragments for a fraction of the cost of currently available methods.
It is easier for a DNA knot...
How can long DNA filaments, which have convoluted and highly knotted structure, manage to pass through the tiny pores of biological systems?
How do metals interact with DNA?
Since a couple of decades, metal-containing drugs have been successfully used to fight against certain types of cancer.
Electrons use DNA like a wire for signaling DNA replication
A Caltech-led study has shown that the electrical wire-like behavior of DNA is involved in the molecule's replication.
Switched-on DNA
DNA, the stuff of life, may very well also pack quite the jolt for engineers trying to advance the development of tiny, low-cost electronic devices.
Researchers are first to see DNA 'blink'
Northwestern University biomedical engineers have developed imaging technology that is the first to see DNA 'blink,' or fluoresce.
Finding our way around DNA
A Salk team developed a tool that maps functional areas of the genome to better understand disease.
A 'strand' of DNA as never before
In a carefully designed polymer, researchers at the Institute of Physical Chemistry of the Polish Academy of Sciences have imprinted a sequence of a single strand of DNA.
Doubling down on DNA
The African clawed frog X. laevis genome contains two full sets of chromosomes from two extinct ancestors.
'Poring over' DNA
Church's team at Harvard's Wyss Institute for Biologically Inspired Engineering and the Harvard Medical School developed a new electronic DNA sequencing platform based on biologically engineered nanopores that could help overcome present limitations.

Related Dna Reading:

Best Science Podcasts 2019

We have hand picked the best science podcasts for 2019. Sit back and enjoy new science podcasts updated daily from your favorite science news services and scientists.
Now Playing: TED Radio Hour

Anthropomorphic
Do animals grieve? Do they have language or consciousness? For a long time, scientists resisted the urge to look for human qualities in animals. This hour, TED speakers explore how that is changing. Guests include biological anthropologist Barbara King, dolphin researcher Denise Herzing, primatologist Frans de Waal, and ecologist Carl Safina.
Now Playing: Science for the People

#SB2 2019 Science Birthday Minisode: Mary Golda Ross
Our second annual Science Birthday is here, and this year we celebrate the wonderful Mary Golda Ross, born 9 August 1908. She died in 2008 at age 99, but left a lasting mark on the science of rocketry and space exploration as an early woman in engineering, and one of the first Native Americans in engineering. Join Rachelle and Bethany for this very special birthday minisode celebrating Mary and her achievements. Thanks to our Patreons who make this show possible! Read more about Mary G. Ross: Interview with Mary Ross on Lash Publications International, by Laurel Sheppard Meet Mary Golda...