Monday, June 29, 2009

Why does DNA testing take so long?

Six months!! But CSI does it in an hour! Why does DNA testing in the real world take so long?

Mostly, because of backlogs and reviews. A given DNA lab can only take in so much work at a time. The biggest limitations are staff, space, and machines. How long does it take to actually process a DNA case after it's been started?

A lab that can do it in 30 days is doing a great job. 60 days is acceptable. This still seems like a long time for one test, doesn't it? Except that it's not one test - it's several tests. And it's not one piece of evidence. To conserve overall time and cost, labs batch together cases and test them simultaneously. (Trust me, without batching, it would take even longer.)

Here's a rough break down of the DNA testing process:

SEROLOGY/SCREENING - An item of evidence first has to be evaluated for the presence of biological fluid. This can involve several tests to detect the presence of semen, saliva, and/or blood. This step is performed on several cases which are then batched together for the remaining steps. It can take 1-2 weeks to create a batch of cases, depending on how much casework the lab processes routinely.

EXTRACTION - Once the areas of suspected biological fluid have been identified, they are cut away from the evidence and put into a test tube. The DNA is then separated from the rest of the cell (and whatever else might be in the sample) using a series of chemical treatments. There are several methods of extraction available in molecular biology. An extraction can take anywhere from 5 minutes to over 2 hours (not including an overnight digestion). Why don't we just use the fastest method? The quality of DNA recovered matters for downstream testing. Poor quality DNA can lead to poor results. Also, since these DNA samples are deposited on items in our surroundings which can contain who knows what kind of inhibitors (did you know that Dr. Pepper inhibits the DNA testing process? So does dirt and certain dyes), it's a good idea to extract the DNA using the highest quality technique available. For our industry, organic extraction is the gold standard.

CLEANING UP - Now that we've got our DNA floating in a tube, we've got to clean it up a bit (for purity). To clean out salts and dyes, the DNA extract is run through a filter, pushing a neutral buffer through the filter along with unwanted impurities while the DNA sits atop the mesh. This step can take up to 2 hours.

QUANTITATION - This step exists purely to provide information for the next step. We need to find out how much DNA we have before we can proceed. So a small portion of the DNA extract (less than 10%) is consumed in a test that measures the amount of DNA present. This quantity is applied to the remaining extract volume to give us an idea of just how much DNA (in a mass measurement, usually in nanograms) is floating around in any given unit of volume (usually in microliters) in the test tube. Setup, processing, analyzing results, and prepping the samples for the next step can take 4-5 hours.

AMPLIFICATION - Polymerase Chain Reaction or PCR is what gives DNA testing its edge. We can start off with DNA from just a few cells and amplify it until we have millions of copies of the segments of DNA we want to looks at. This process mimics the cell's natural DNA duplication technique. Enzyme, nucleotides, and commerically made DNA primers are thrown into a fresh test tube along with a portion of the DNA extract (the quantity of extract added is determined by the quantitation step). Several "loci" (or specific pieces of DNA) are copied during a series of heat and cool cycles that activates the enzyme. We don't copy the entire DNA strand - just the pieces we want to test for. How many pieces? The FBI has identified 13 core loci that are used throughout the field. Some commercial kits throw in primers for a couple more just for good measure. Setup and processing takes at least 4 hours.

DETECTION - Now we have to separate out our DNA copies so we can "read" them. Imagine you're reading a book. You turn the page, but instead of the words being aligned in nice rows so you can read them, they are all superimposed on top of one another. How are you to figure out what's going on? It's the same with our DNA copies. In the test tube, they are all floating around together. To read them, we run them through a sieving polymer that separates the DNA pieces based on sized. We can then look at each piece of DNA and interpret it into a DNA profile. Depending on your equipment, this process can take up to 48 hours for a batch, but most modern labs can complete this in under 6 hours.

ANALYSIS - This is where I could just kill the writers of CSI. On that show, they put the DNA into a machine, and out pops a DNA profile. Sorry, but it doesn't work that way in the real world. As with any data, you have to analyze it first. DNA data looks a lot like a piece of paper from a polygraph machine. There's a baseline that's continuous, and DNA shows up as a peak. We have specialized software that helps us to figure out what each peak of DNA corresponds to in terms of our DNA profile, but sometimes we get peaks that are not true DNA. Analysts are trained to spot these anomalies and discount them from the profile. An analyst must understand the mechanics of PCR and of DNA separation and detection to know what's DNA and what's not. Depending on the number of items of evidence in a case, analysis can take up to an hour or more.

INTERPRETATION AND REPORTING - Once the DNA profiles have been generated, the DNA analyst compares the evidence items to the reference samples (samples from known victims or suspects) to see if any matches or exclusions can be claimed. If there is a match, statistical calculations must be performed. If there are mixed results (i.e. a door knob is likely to contain DNA from more than one person), the analyst must see if a major donor can be discerned. If it cannot, then the analyst must see if the reference DNA profiles could be included as possible contributors to the mixture. An inclusion would require another set of statistical calculations to be performed (how many other people in the general population could also have contributed to this mixture?). This process can be very time consuming depending on how complicated the data obtained is. Analyst often times need to consult with each other to arrive at a conclusion that is scientifically sound. (Oh, and by the way, Forensic DNA reports are extremely difficult to read because we're making sure that they're scientifically sound. We can't just say "he did it.") This step can take up to 4 hours for a complicated case. Only an hour for an easy case.

REVIEW - This step takes the longest - days to weeks. Why? Because other analyst who did not work the case have to find time in their day to review the work of a colleague. Usually a supervisor or technical leader also has to review it. Sometimes a crime lab director has to sign off on the results as well. With smaller labs, there is limited personnel available for these reviews. It may sit on a desk for days or weeks before a review can be done.

Finally, we're done. And none of this included any rework. Rework occurs when the data doesn't look as good as it could. To aid interpretation, the sample is reprocessed (starting as far back as extraction sometimes, but usually only starting at amplification or detection). Whether or not the sample needs to be reprocessed cannot be determined until after the initial detection step in most labs.

Without reprocessing, the above steps add up to 24 hours not including the 1-2 weeks at the beginning to build a batch (see serology above) and not including the days or weeks it takes to complete the review process.

But will it always be like this? Research is underway (and quite far along, actually) for the development of a system that will process DNA samples in 4 hours. However, this system is not for tricky DNA samples. It will only be applicable to very simple run-of-the-mill DNA (like heavy blood stains).

Until then, please be patient with your DNA analysts. We are trying as hard as we can to finish our work. Some extra-busy labs even divide their work force into shifts so that a case can be worked on for more than 8 hours a day.

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