Twenty six years ago when I finished medical school I knew a little bit about DNA—about its structure and how it interacts with messenger RNA to build proteins and, ultimately, everything else. In the following years, I started using a few laboratory tests that involved DNA. In the workup of autoimmune diseases, I could order anti-double-stranded DNA assays. When polymerase chain reaction (PCR) tests became available for Hepatitis C and HIV, I used them. But my level of understanding was pretty basic: I knew when to order the test and how to interpret the results, but I really had no idea what actually went on in a DNA lab.

On those rare occasions that I visit a clinical laboratory, it’s almost always to see why a stat lab is taking so much time. A result may mean the difference between admitting someone to the hospital or following him at home. It’s always rewarding to make these visits because I get to know the lab technicians face to-face and I gain a little more appreciation for the challenges of their jobs.

DNA testing—specifically, DNA “fingerprinting”–is not done in our hospital lab. There are good reasons for this. First, we couldn’t afford it and next a hospital or clinic is a bad place to do DNA testing since the potential for contaminating a sample is very high.

My first requirement before entering the DNA lab was to have my own cheeks swabbed for a DNA analysis just in case I inadvertently contaminated something.

The sample I observed arrived in a FedEx package sent by an attorney from out-of-state. The sample was from a client he was representing in a criminal case. I have no idea what actual legal issues were involved. The lab’s task was simply to characterize the DNA and report back to the attorney.

At every step of the process, the scientist doing the analysis makes an entry in a log to show an unbroken chain of custody of the sample and to document every step in the actual analysis. In this case, the scientist is my son, Jason Eshleman, Ph.D., senior research scientist for Trace Genetics, a laboratory specializing in specialty DNA analysis.

Step one in the analysis is getting the DNA off of the swab. This involves using a lysis buffer—a kind of detergent–that breaks down the cheek cells and releases DNA and other molecules into a solution. Next, depending on the precision required, the DNA may be purified, but in this case it wasn’t necessary.

Next in the Polymerase Chain Reaction (PCR) phase. This is an elegant bit of applied biochemistry that richly deserved the 1993 Nobel Prize in chemistry it won for Kary Mullis. PCR as practiced today requires a machine called a thermal cycler. The thermal cycler cooks the DNA sample at 95º C, causing the strand to unwind and split into two single-stranded chains. The process is called denaturation.

Next the mixture is cooled to somewhere between 50 and 65º C to allow DNA primers to attach (the process is called annealing) to the portion of DNA being studied. A DNA primer is a short, artificial strand of DNA that attaches to known loci on a DNA strand.

In the case of the forensic analysis I observed, the primers were attached (annealed) at opposite ends of two hypervariable regions of the DNA. Hypervariable regions are just that: they are sequences of perhaps a 1000 base pairs that apparently code for nothing, and which are so variable that the possibility of two individuals having the same sequence is vanishingly small.

Next comes what is called the extension phase. After the primers have attached, the mixture is heated to about 70º C and a DNA polymerase enzyme known a T.aq and a mixture of free nucleotides are added. T.aq is short for Thermus aquaticus. T.aq. is a bacterium that lives in hot springs. It lives (and its enzymes function) at temperatures that would kill most living things. Starting at one primer, the enzyme catalyzes the formation of a complementary copy of the single DNA strands. T.aq. is a very expensive molecule.

There follows a repetitive cycling of temperatures. The mixture is again heated to 90º C, the DNA is again denatured, and then the mixture is cooled to allow the copies to be made. This process is repeated from 25 to 45 times. If the process were perfect, this would create somewhere between 2 to the 25th and 2 to the 45th power. Two to the 25th is more than 32 million. Although less than perfect, this process creates a huge number of identical strands of DNA! This makes the next step much easier. The Wikipedia discussion of PCR has some nice diagrams that may help you understand the process.

I’ll discuss the next steps in the analysis in another posting. In the meantime, take a look at the posting that follows that includes a picture of an actual DNA microarray used in the analytic process.

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