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March 2002
Vol. 5, No. 3, pp 25–26.
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High-throughput DNA purification

Automation is the buzz that’s busting bottlenecks in large-scale population studies
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ILLUSTRATION: TONY FERNANDEZ

With so much genomics research going on in the United States, it is easy to miss the hum of initiatives around the world. Researchers eager to lay hands on genes controlling disease have launched genomics projects in lands as distant and different as Iceland, Estonia, Sardinia, Newfoundland, and the South Pacific island kingdom of Tonga. Together, these population projects account for millions of large-volume patient samples, yet the idea of purifying DNA from even a few thousand samples remains a daunting prospect.

Scientists want to use automated genotyping to examine thousands of gene variants in millions of people, tracking down single genes controlling the rarest of diseases and locating the many genes that subtly influence our most widespread and complex diseases. Genomics in a faraway place can make those tasks easier: Quirks in isolated gene pools sometimes give researchers piercing insight into the genetic aspects of complex diseases. Genomic research in Icelandic populations has already resulted in the identification of genes linked to obesity, anxiety, diabetes, schizophrenia, Parkinson’s disease, and rheumatoid arthritis.

Genomics is also spreading for other reasons. Iceland’s genomics project is aided by family trees that trace as far back as the 9th century, when ties of blood were recorded with the ink of blood, and animal skins served in place of paper. Estonia drives its project forward on sheer ambition. Its goal is to own the world’s largest genome project within five years, holding DNA samples from 1 million people—2 of every 3 Estonians.

The buzz behind this genetic land rush is that population geneticists must find faster methods than preparing human genomic DNA by hand. Think of it this way. Which of the following phrases sounds out of place in a genomics laboratory?

  • Thousands of bases sequenced per day.
  • Thousands of genotypes per day.
  • Dozens of manual DNA preps per day.

The fact is that population genomics requires automation wherever possible, including DNA purification. Manual preps are fine for small projects, but competing in genomics by using hand-prepped DNA is like filling the tank at a NASCAR pit stop by using a teaspoon.

First to Finnish
One researcher who understands this is Markus Perola, who conducts genomics research in the human genetics laboratory of Leena Peltonen at the University of California, Los Angeles (UCLA). While working with Peltonen as a graduate student at the National Public Health Institute in Helsinki, Perola studied rare recessive disorders in Finnish families, mapping and isolating the responsible genes. “Many people call Finland ideal for studying genetic disorders,” Perola comments, “because we are sort of isolated, separated for centuries without much immigration.”

Perola was given a thesis project on the genetics of hypertension. The complexity of the disease required DNA samples from many people, and he quickly became expert at isolating and purifying genomic DNA. Around the same time, epidemiologists at the institute began studies calling for enormous numbers of DNA samples. Perola was enlisted to help them. Soon, he found himself acting as the head of a team of eight, preparing DNA from 60,000 Finns entirely by hand, using the old phenol–chloroform method (see box, “DNA preparation: Old and new”).

After receiving his doctorate, Perola followed Peltonen to her new laboratory in California, where he began using DNA microarrays to study the genetics of psychiatric and cardiovascular problems. Soon after his arrival, UCLA’s human genetics department asked him to advise it on the best way to handle DNA preparation. He never doubted that the answer was to automate. The question was how.

The standard source of human DNA is blood drawn in 10-mL aliquots. Perola needed an instrument that would work with minimal human intervention, extracting DNA pure enough to store for years without degradation. Ideally, the preparation chemistry would not generate toxic wastes. He decided on the Autopure LS, introduced in 2000 by Gentra Systems, Inc. (Minneapolis; www.gentra.com).

Archival-quality DNA
It should be possible to put genomic DNA in cold storage and forget about it, without wondering if it will be there when you need it. But as Perola knows, unless DNA is prepared correctly it will not be of archival quality, and researchers’ hopes of meaningful findings will fragment and degrade along with their precious DNA samples. For even in the cold, incorrectly prepared DNA can break down.

DNA preps degrade for three reasons. They can be contaminated by bacteria, but storage at 4 ºC or lower prevents this, says Perola. More often, degradation is due to contamination of the sample with DNase from skin. Wearing gloves while preparing DNA can solve this. Generally overlooked, however, is degradation caused by continual cycles of freezing and thawing. Perola minimizes the problem by planning ahead.

“We have a one-usage tube, as we call it, for aliquoting,” he says, “and a stock tube that is used once every few years.” As an extra precaution, each sample has two stock tubes, which he stores at –20 ºC. A better temperature would be –80 ºC, which avoids slow evaporation and changes in DNA concentration, but freezing at –20 ºC is less expensive.

Perola strongly advises preparing DNA as soon as possible after drawing blood. Storing blood first and preparing DNA later sounds convenient but creates unnecessary work. His experience is consistent with findings that Gentra has published for some years: Storing blood results in lower yields (often substantially lower), no matter what the purification method.

Productivity
UCLA’s human genetics department organizes genomics with core laboratories. Instead of each faculty member’s laboratory doing its own genotyping, sequencing, and DNA extraction, core laboratories boost department productivity by handling these functions as services. UCLA already has core laboratories for genotyping and sequencing. Perola set up an automated DNA extraction service before returning to Finland, where he will continue his work in human genetics.

What Perola did for UCLA, he hopes to repeat in his laboratory back home. He sees the difference that automation makes. “At UCLA, one person can do about all that needs to be done,” says Perola. “The tech puts the samples on and comes back later. There’s the DNA.” Another bonus is not having to worry about phenol and chloroform. “This is important for us because both phenol and chloroform are carcinogenic and toxic,” he says. “And they also break DNA.”

Frozen treasure
Recently, Perola found occasion to reuse DNA samples frozen five years earlier. He and Peltonen decided to reanalyze genomics data from earlier studies on Finnish families with hypertension, obesity, osteoarthritis, migraine, and familial combined hyperlipidemia. This time, they hoped that the data could be used to pinpoint genes controlling traits unrelated to those diseases. Fortunately, a great deal of patient information was collected with each blood sample used for DNA purification, giving the researchers the opportunity to use the samples retrospectively for analysis of linkage to other diseases and traits. Soon, they found something. But to be sure of what they had, they needed to retrieve the stored DNA samples and genotype them one more time.

“That’s an example of the kind of treasure you have once you have these materials,” Perola says with enthusiasm. “One analysis is not enough. You have to reanalyze in order to get all the information out.” The discovery confirmed by their return to the archives—that a gene on chromosome 7 is linked to height—was published in the July 2000 issue of the American Journal of Human Genetics.

With the help of heavy-duty nucleic acid purification systems that can produce archival-quality DNA, investigators like Markus Perola can look forward to more successes. Each one is a part of the quiet hum of progress in international research, which is punctuated occasionally by the sound of a geneticist busting through another bottleneck. And in genomics research, busting barriers is the buzz.


Darin O’Brien is a product manager at Gentra Systems, Inc. Send your comments or questions regarding this article to mdd@acs.org or the Editorial Office by fax at 202-776-8166 or by post at 1155 16th Street, NW; Washington, DC 20036.

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