Daily Archives: January 11, 2013

BERKELEY A simple, precise and inexpensive method for cutting DNA to insert genes into human cells could transform genetic medicine, making routine what now are expensive, complicated and rare procedures for replacing defective genes in order to fix genetic disease or even cure AIDS.

Discovered last year by Jennifer Doudna and Martin Jinek of the Howard Hughes Medical Institute and University of California, Berkeley, and Emmanuelle Charpentier of the Laboratory for Molecular Infection Medicine-Sweden, the technique was labeled a "tour de force" in a 2012 review in the journal Nature Biotechnology.

That review was based solely on the team's June 28, 2012, Science paper, in which the researchers described a new method of precisely targeting and cutting DNA in bacteria.

Two new papers published last week in the journal Science Express demonstrate that the technique also works in human cells. A paper by Doudna and her team reporting similarly successful results in human cells has been accepted for publication by the new open-access journal eLife.

"The ability to modify specific elements of an organism's genes has been essential to advance our understanding of biology, including human health," said Doudna, a professor of molecular and cell biology and of chemistry and a Howard Hughes Medical Institute Investigator at UC Berkeley. "However, the techniques for making these modifications in animals and humans have been a huge bottleneck in both research and the development of human therapeutics.

"This is going to remove a major bottleneck in the field, because it means that essentially anybody can use this kind of genome editing or reprogramming to introduce genetic changes into mammalian or, quite likely, other eukaryotic systems."

"I think this is going to be a real hit," said George Church, professor of genetics at Harvard Medical School and principal author of one of the Science Express papers. "There are going to be a lot of people practicing this method because it is easier and about 100 times more compact than other techniques."

"Based on the feedback we've received, it's possible that this technique will completely revolutionize genome engineering in animals and plants," said Doudna, who also holds an appointment at Lawrence Berkeley National Laboratory. "It's easy to program and could potentially be as powerful as the Polymerase Chain Reaction (PCR)."

The latter technique made it easy to generate millions of copies of small pieces of DNA and permanently altered biological research and medical genetics.

Two developments - zinc-finger nucleases and TALEN (Transcription Activator-Like Effector Nucleases) proteins have gotten a lot of attention recently, including being together named one of the top 10 scientific breakthroughs of 2012 by Science magazine. The magazine labeled them "cruise missiles" because both techniques allow researchers to home in on a particular part of a genome and snip the double-stranded DNA there and there only.

More:
DNA snipping technique could transform gene therapy

DNA is a linear information storage medium, like a magnetic tape, but written with a very limited character setjust A, C, G, and T.

Heres how we read that tape today: we dub a copy of the tape, stop at some point when we reach the character A, cut the tape there, and toss the fragment into the A bin. Then we make another copy, stopping at some point on another A, snip the new copy short, and toss that fragment into the bin. We repeat this millions of times, and then start over again for C. And again for G and T. When were done, we measure lengths of all of the millions of A fragments, C fragments, G fragments, and T fragments and then use a computer to sort them by length and tell us where in the sequence we encountered all of the As, Cs, Gs, and Ts. What could go wrong?

It would obviously be much quicker, cheaper, and more reliable if we had a DNA-read-head, a device we could just run the DNA-tape over to detect the sequence directly, in a single pass. Back in 1995, George Church and colleagues figured out one approach: it might be possible to use a low intensity electric current to pull long strands of DNA through nanometer-scale pores in a membrane and measure the electric field variations of the four nucleic acidsA, C, G, Tas they passed through.

Scientists are working on it, but were not there yet. Key questions remain unanswered. One of them is fundamental: How does DNA move through a pore? Does it slide through end-on? Or does the pore grab it somewhere in between, bend it double, and suck it through doubled over (like a strand of spaghetti slurped up from the middle). Every cell in your body, after all, carries about two or three meters of DNA. Even a mere fragment of 50,000 base pairs is about 16.5 micrometers longthousands of times the diameter of a nanopore. If mere chance determines the orientation, one would expect that almost all DNA should pass through a nanopore doubled over.

Fortunately for the future of bedside genomics, that doesnt seem to be the case. Researchers at Brown University have developed an elegant method for determining exactly how a DNA molecules passes through a nanopore. They can see if it slips through end-on or goes through doubled overand, if it does double over, they can tell where along its length it folds.

Physicists Mirna Mihovilovic, Nick Hagerty, and Derek Stein followed the travels of about 1100 pieces of double-stranded DNA (for aficionados, they were 48,502 base-pair segments of bacteriophage lambda, measuring about 16.5 micrometers in length) as they slithered through an 8-nanometer-diameter pore in a 20-nm-thick silicon nitride membrane, urged along by a 3.6 nanoampere current. They measured the current flowing through the pore 50,000 times a second, and found it dropped when passing DNA partially blocked the pore. The interruptions lasted only a couple of milliseconds, and the magnitude of the current reduction was proportional to the cross-section of DNA blocking the porewhich is to say that the current dropped about 0.28 nanoamperes when there was a single strand in the pore, and about 0.56 nA when the DNA passed through sideways. Thus, the current profile revealed the DNAs orientation: the relative durations of the double-strand and single-strand current reductions showed just where the molecule had folded. (So a 3-millisecond drop of 0.28 nA might indicate that the DNA had speared straight through the pore, while a 1.5-millisecond drop of 0.56 nA indicated a fold exactly in the middle. The diagram at right makes it clearer: ECD stands for event charge deficit, the current drop integrated over time, which remains approximately constant for each DNA passage.)

The researchers found that DNA passes through the pore smoothly, end-on, about 25% of the timefar more than most current models would predict. This surprisingly high proportionindicating, they say that the orientation is a function of the configurational entropy of the approaching polymerbodes well for developing a nanopore-based DNA direct reader.

Figures:Derek Stein/Brown University

Continued here:
Following DNA Down the Nanopore Rabbit Hole

Jan. 10, 2013 The first rigorous analysis of the crime-fighting power of DNA profiling finds substantial evidence of its effectiveness.

According to the study from the University of Virginia, violent offenders whose DNA is collected and stored in a database are 23.4 percent more likely to be convicted of another crime within three years than their unprofiled counterparts. In other words, profiled offenders, especially those under age 25 and those with multiple convictions, continue to commit new offenses, but are caught much more often than those not in the database.

DNA databases reduce crime rates, especially in categories where forensic evidence is likely to be collected at the scene -- murder, rape, assault and vehicle theft.

Estimates of the marginal cost of preventing each crime suggest that DNA databases are orders of magnitude more cost-effective than alternatives like hiring police or locking people up longer.

Though it may come as a surprise to viewers of the popular TV show "CSI" or professionals who work in the criminal justice system, this is the first rigorous analysis of the crime-fighting benefits of DNA profiling, said the study's author, Jennifer Doleac, an assistant professor of public policy and economics at U.Va.'s Frank Batten School of Leadership and Public Policy.

The study, "The Effects of DNA Databases on Crime," is published in the Batten School's new working paper series, and currently under review at a peer-reviewed journal.

Since 1988, every U.S. state has established a database of criminal offenders' DNA profiles, and these databases have been periodically expanded -- for instance, to include individuals convicted of an additional type of felony. Such expansions are often in response to widely publicized "if only" cases where terrible crimes could have been prevented if only a particular offender had been required to submit a DNA sample based on a previous conviction, Doleac said.

She used state database expansion events between 1988 and 2008 to compare when very similar offenders were released from prison just a few weeks apart -- some before the effective date of the DNA database expansion and others afterward. Crucially, in all other characteristics that might affect recidivism risk, the offenders were a homogenous set, so any subsequent differences between the two groups could be attributed to the effect of DNA profiling.

Drawing on corrections department administrative data from seven states, Doleac assembled a data set of 3,949 offenders. Of those, 1,993 were released before before a database expansion (the control group) and 1,956 were released afterward.

On average, Doleac found that DNA profiling does help identify suspects, just as proponents have assumed. Profiled offenders are 23.4 percent more likely to be convicted of another crime within three years than their unprofiled counterparts.

Go here to read the rest:
First cost-benefit analysis of DNA profiling vindicates 'CSI' fans

Fetal DNA circulating in a pregnant mothers blood can be used to detect a wide variety of genetic abnormalities before birth, opening the door for noninvasive testing for more conditions.

By sequencing DNA that escapes into womens bloodstreams, scientists were able to detect disease-causing mutations that are now normally found by piercing a mothers womb with a needle to get amniotic fluid, according to a study in the American Journal of Human Genetics.

Amniocentesis, the standard procedure for prenatally testing for genetic conditions such as Down syndrome, carries a low risk of miscarriage. Obtaining DNA from a blood sample from the mother carries virtually no risk, and may enable doctors to expand their reach and accuracy as they look for genetic disease, said Cynthia Morton, a Harvard Medical School geneticist who performs prenatal tests at Brigham & Womens Hospital in Boston.

This could largely replace invasive testing, she said in a telephone interview, and, no doubt, is an exciting next step in the future of prenatal testing.

The study was done by scientists at Tufts Medical Center in Boston and Verinata Health Inc. in Redwood City, California. Illumina Inc. (ILMN), the biggest maker of DNA sequencers, said this week that it will buy Verinata for $350 million plus as much as $100 million in milestone payments through 2015.

Interest in sequencing fetuses and newborns is increasing as more laboratories are showing that DNA analysis can quickly diagnose rare diseases that once took years to unravel. The U.S. National Human Genome Research Institute and the National Institute of Child Health and Newborn Development have set aside $25 million to study questions related to sequencing newborns over the next five years.

Verinata and other companies already offer blood tests that analyze circulating fetal DNA to diagnose Down syndrome, a genetic condition in which a baby is born with three copies of chromosome 21, instead of the normal two. The same tests can detect other conditions in which the fetus has too many copies of certain chromosomes, which are the packages that hold large amounts of DNA within the cells nucleus.

In the study published today, the team showed that they can detect far smaller genetic flaws that affect just portions of chromosomes. The test was able to find abnormalities involving as little as 100 kilobases of DNA, a fraction of the millions of chemical bases that each chromosome normally contains.

The price of sequencing DNA is falling quickly, and as it does, the scientists are using the procedure to replace and expand on established medical tests. In a study released yesterday in the journal Science Translational Medicine, for example, researchers showed that DNA analysis of the Pap smear for cervical cancer can also identify malignancies of the ovaries and endometrium.

Currently, doctors who believe a fetus may harbor a genetic condition analyze chromosomes and DNA taken by amniocentesis.

Original post:
DNA in Mother’s Blood Can Spot Genetic Mutations in Fetus