Science Education

The Interrogation of Mr. DNA, Part 2 of 3: Amplification

Previously, on The Interrogation of Mr. DNA:

A woman, found dead in her apartment. Blood on her mouth and teeth which may belong to the murderer. At the crime lab, our heroic technician, Greg, has just finished extracting the DNA from the blood spots. No pee breaks on this one, folks. Our show resumes.

With a clean DNA sample from the victim’s mouth and another sample from her wound, Greg is ready to compare and analyze. But compare what to what?

Throughout our genome are littered these short tandem repeats, or STRs, which are composed of repeated motifs of two, three, four, five, or six DNA bases. A two-nucleotide STR might be a repeat of the motif “CA”, which can be particularly funny in French-speaking countries. A three-nucleotide STR can be a repeat of the motif “TAG”, and so on. Because these stretches are repetitive (“CACACACACACA” as opposed to “TTGACGATCCGATG”, for example), the protein tasked with replicating the DNA has a hard time keeping track of where it has been and which bases it has yet to replicate. “Did I do this ‘CA’ already or not?” it might ask itself if it were sentient. Because of this hypnotic issue, the protein sometimes slips… and makes a mistake.

This is how we go from Daddy having 12 of these “CA”s in a row to Sonny-boy having 13.

Now multiply this situation by 2.8 million STRs in a genome and you will get an idea of the potential variability between individuals1.

The Federal Bureau of Investigation (FBI) chose 13 of these STRs, spread all over the genome on many different chromosomes, as its standard panel for forensic testing. This is what Greg has to do, now: test his DNA samples to determine the length of these 13 repeats and see if there is a match.

In the early 1980s, a technique known as the polymerase chain reaction (or PCR) was developed which revolutionized molecular biology, genetics, and forensic biology. What it allowed scientists to do is to figuratively “photocopy” a specific part of the genome to such a high amount that its detection was improved. Without this amplification, Greg would require a lot of DNA to get a clean signal that would allow him to state, with “legal” certainty, that such a repeat is of a certain size. Given the fact that Greg works with dried blood spots, semen stains on jeans, and a few hairs recovered from crime scenes, he is very happy to have at his disposal a technique like PCR that requires very little DNA to work.

PCR is simple: it’s selective replication. Scientists learned to harness the power of the machinery that naturally replicates our own DNA and to direct it toward their region of interest. The reaction is performed as a cycle of three steps:

1. The DNA helix, which is made up of two strands, must be denatured. This means that the links between these two strands, which are complements to each other, must be temporarily broken. This is accomplished in the lab by turning the temperature up to near-boiling.

2. As the temperature is brought down, short pieces of DNA called primers, designed by scientists to bind the DNA before and after their region of interest, anneal to the DNA, thus creating very short stretches of double-stranded DNA.

3. As the temperature rises again, the protein responsible for replicating DNA binds these short stretches of double-stranded DNA and elongates them, creating brand-new DNA that complements the single-stranded template.

We then return to step 1 and cycle through these steps between 25 and 40 times on average, until we end up with a billion copies of our region of interest.

This may be a bit difficult to understand at first. Fear not, for the amazing people at the Howard Hughes Medical Institute (HHMI) have put together this brilliant computer-generated animation of what PCR looks like from the DNA molecule’s point-of-view.

Reproducing what our bodies do in test tubes is not as simple as I made it out to be. For instance, in the body, the DNA molecule is partly denatured by proteins called helicases and topoisomerases. In the lab, Greg has to turn the heat up to 95ºC to achieve the same result which, as it turns out, is a mood killer for the protein used to replicate the DNA, the so-called DNA polymerase. The way this problem was solved was by ingeniously collecting bacteria that live in extreme environments—microorganisms like Thermus aquaticus which thrive in hot springs and thermal vents—and isolating their DNA polymerase, which has evolved to withstand these high temperatures.

Luckily for Greg, all the reagents he needs to conduct this PCR amplification on his DNA samples—the primers, the polymerase, the salts and buffering solutions—come in a nifty little kit that biotechnology companies sell. He can thus amplify all 13 repeats in a single reaction, a concept known as a “multiplex”. When all the ingredients have been added to a small reaction tube, Greg puts the tube in a special kind of oven called a “thermal cycler”. The cycler is a piece of machinery which tightly controls the temperature of its heating block. It can thus be programmed to achieve certain temperatures very quickly and hold them for the required amount of time. The thermal protocol programmed into the machine is the PCR cyclic method described above: a series of denaturations, annealings, and elongations. At the end of the reaction, the contents of the tubes may not look any different, but each tube is filled with a billion of these short stretches of DNA containing these repeats.

Now Greg is faced with a problem. How can he measure the lengths of these repeats?

(Feature picture by Brandon Anderson)

1. Payseur BA, Jing P, Haasl RJ. 2011. “A genomic portrait of human microsatellite variation”. Mol Biol Evol 28(1): 303-12.


5 thoughts on “The Interrogation of Mr. DNA, Part 2 of 3: Amplification

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