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.
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
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.
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
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.
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."