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The Little Things that Make a Big Difference
CHORI Researchers Develop New DNA Extraction Technique

A recent publication by CHORI scientist Janelle Noble, PhD, and her colleague Julie Lane, BS, in the Journal of Diabetes Science and Technology report on a few simple steps Dr. Noble developed to improve the amount of DNA that can be extracted for research analyses from Guthrie cards – the newborn blood spots collected right after birth for standard neonatal screening.

“We didn’t do anything conceptually brilliant here, but because Guthrie cards are such a valuable resource, and such a limited resource, you really want to get every last molecule out of them that you can,” says Dr. Noble.
“Because Guthrie cards are such a valuable resource, and such a limited resource, you really want to get every last molecule out of them that you can.”

Guthrie cards are collected at birth – a series of five blood spots taken from newborns with a heel-prick that are used to screen for certain conditions or diseases that can be life-threatening when not treated immediately. Beyond these state-mandated neonatal screenings, the Guthrie cards have no specific use, and are stored for a number of years before being destroyed. These innocuous drops of blood, however, can be an incredible research asset.
“For researchers doing population-based studies who want to look at how ethnicity impacts genetics and health, the Guthrie cards can be an invaluable tool.”
“In this case, we received permission from the State of California, Genetic Disease Branch to use the cards in our human leukocyte antigen – or HLA – studies,” says Dr. Noble.

Dr. Noble used the Guthrie cards to help identify HLA patterns in African Americans that could reveal important, ethnic-specific information about the genetics of HLA-associated diseases, like type 1 diabetes. The biggest problem with Guthrie cards, however, is that they provide only five little spots of blood from which DNA can be extracted.

“When we do HLA genotyping, we’re looking at a large number of different genes for their frequency in different populations. This requires 6 to 8 assays of the same DNA to do a complete genotyping,” explains Dr. Noble.
“Using standard methods for extracting DNA from Guthrie cards, we would have run out after just 3 to 5 of the assays.”

And because the Guthrie cards are a non-renewable resource, running out of DNA means the premature closure of a study.

As Dr. Noble explains, “We know only that the blood comes from infants born in a certain year in California and their ethnicity based on their parents self-reporting. Unlike a consented study, where you can get more blood samples from the same patient, we don’t know who any of these people are. This makes the Guthrie cards a precious resource.”

As with Dr. Noble’s study, maximizing the amount of DNA extracted from these tiny little blood spots can be the difference between completing a successful study or not. Which was why Dr. Noble did a little research on DNA extraction methods.

“We wanted to do a review of the literature, to make sure we were doing the extraction in the best way possible, and discovered that there just wasn’t really a lot of information out there,” says Dr. Noble.

As a result, Dr. Noble and her colleagues took matters into their own hands. They started with a slight modification of the standard protocol for DNA extraction – the utilization of a new-to-the-market reagent. The addition of the reagent helped, in particular with the first step of getting the blood itself out of the filter papers, but what really made the critical difference was the addition of one simple step.
“Normally, when you extract the DNA like this, you bind the DNA to a silica filter, you wash away the other particles you don’t want, and then you add what is called elution buffer to detach the DNA for analysis,” explains Dr. Noble.

“We simply pre-heat the elution buffer. We make everything nice and warm, and soak it for a little bit longer than normal. We find that we get a much greater yield.”

A ten-fold increase in the amount of extracted DNA, to be exact.

“It’s kind of like doing your laundry or dishes in warm water instead of cold,” says Dr. Noble.
“What we did is nothing earth-shattering, but it makes a big difference in what you can do with this very limited source of DNA.”
A simple modification to be sure, but one that provided ten times as much DNA, allowing Dr. Noble and her colleagues to successfully complete their HLA analysis.

“What we did is nothing earth-shattering, but it makes a big difference in what you can do with this very limited source of DNA,” says Dr. Noble. “We thought it was important to share the benefits of these minor modifications, so when someone else is in just the same position in which found ourselves, they can do a literature search on increasing DNA yields and actually find an answer.”

Thanks to Dr. Noble’s efforts, researchers will be able to do just that.

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Tuesday, May 17, 2011 8:19 AM

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