How Are Microsatellite Repeats Counted?

So
How Do They Count Repeats?
Anyway?

Once a segment of DNA has been singled out by its special primers and amplified by PCR the number of motifs (repeats) of the target STR can be easily determined by gel electrophoresis. The mix of billions of short fragments from the PCR is loaded into either a shallow tray or a series of glass capillary tubes that contain a gel solution that serves as a sieving matrix. During electrophoresis, a voltage is created across the gel so that one end is made positive and the other negative. Since DNA is slightly negative, its fragments will move to the positive end of the gel. The mobility of a piece of DNA in the gel is determined by its size. If a mixture containing fragments of all the known range of repeats is run through the gel in a "lane" next to the sample, the fragments will spread out according to length forming what is known as a allelic ladder. The length in base pairs can then be determined by comparison.

Multiplex Reactions

To save time, money, and materials, much work has gone into developing procedures whereby test DNA can be "incubated" with a combination of PCR primers for several different markers at one time. Primers are designed to bind at only one spot in the genome so that during the PCR each primer should be busily dissecting out and amplifying its own marker exclusively.

The task of sorting out the results at the end is facilitated by adding various colored fluorescent tags to the fragments. Fluorescent labeling of DNA fragments may be performed in several ways. The most common method is to incorporate a fluorescent dye on the 5'-end of a PCR primer so that during PCR amplification either the forward or the reverse strand of DNA will be labeled.

At the end of the PCR, the mixture will be run through a gel electrophoresis slab or an array of microtubules each filled with a gel. Since each amplified fragment has a discrete length that depends on the number of repeat motifs, it will move through the gel at a different rate. The colors of the fluorescent labels are coordinated with the size ranges of the markers so that several markers in the same size range can be run together, each labeled with a different color.

The number above each peak in the figure above is the DYS marker number for the fragment. The X axis represents the PCR Product Size i.e. number of base pairs in the fragment. Thus the smallest fragments are DYS 426 in the upper trace and DYS391 in the lower trace.

By comparing with the mobility rates of fragments of known lengths, it is possible to determine the length in base pairs for each marker and thus by calculation, the number of repeats which is the number that you will receive in your report.

In the tracing above, four different fluorescent labels have been used--green, blue, red, and black (which was probably actually yellow which does not show up very well.) Two different sets of markers are shown. The upper tracing represents the results of multiplexing 19 different markers--each labeled by its DYS identification number. The bottom tracing shows the results of nine of the markers in the upper trace. Notice, for example, how the markers DYS389I and DYS388 are only distinguishable as two separate markers because of their different colored fluorescence.

Also note that the height of the fluorescence peak for each marker is not an important value as only primers are labeled--one per fragment no matter how long or short the fragment. The location of the peak on the x axis represents the number of base pairs in the fragment which is a function of the number of repeats. Basically, once the number of base pairs in the fragment is determined, it is a matter of subtracting the number of base pairs in the primer from the total length then dividing that number by the number of bps that make up on marker motif. This is a bit of an oversimplification because some markers may be made of more than one repeating pattern and some primers may actually be part of the actual STR region. Nevertheless, the concept is the same. All measurements and calculations are done by computer.

In most large labs, an instrument called an automated DNA sequencer is used to run the gels and record the different colors. There's an ultraviolet laser built into the machine that shoots through the gel near the bottom and scans side to side, checking for bands of fluorescent colors to pass through its beam. It is possible to run as many as 96 samples through the gel at one time! The ABI PRISM® 3700 DNA Analyzer shown at the right is, according to the Applied Biosystems website, "a fully automated, multi-capillary electrophoresis instrument designed for use in production-scale DNA analysis. It can automatically analyze multiple runs of 96 samples, which enables 24-hour unattended operation."

If you really want to get into reading about multiplexing, there is a detailed protocol (set of directions) at http://www.ihwg.org/protocols/hct/HCT-microsatellite-protocol.pdf which may give you some insight into the activities that are going on in a DNA typing lab. It's pretty technical but if you've made it this far, you should be able to follow some of it. I had a sixth grade teacher who said if you come to a word you don't know just call it "horse" and keep going. You'll probably get something out of it.

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