Biology Reference
In-Depth Information
solution to separate the DNA molecules [33 - 37]. Applied Biosystems provide the
most commonly used CE systems and all these have multicolour detection capacity.
The ABI PRISM
310 Genetic Analyzer has a single capillary and analyses up to
48 samples per day; the ABI PRISM
3100, 3130
xl
and 3500 Genetic Analyzers,
which have up to 16 capillaries and can analyse over 1000 samples per day, and the
ABI PRISM
3700 and Applied Biosystems 3730
xl
Genetic Analyzers, which can
have up to 96 capillaries and can analyse over 4000 samples per day.
Before electrophoresis, the PCR sample is prepared by mixing approximately 1
l
of the reaction with 10 - 20 µl of deionized formamide. The internal-lane size standard
is also added at this point. The deionized formamide denatures the DNA; heat-
ing the samples to 95
◦
C is routinely carried out to ensure that the PCR products
are single-stranded. The samples are transferred into the capillary using electroki-
netic injection, a voltage is applied and charged molecules, including the amplified
DNA fragments and the internal lane size standards, migrate into the capillary.
After injection, a constant voltage is applied across the capillary and the PCR prod-
ucts migrate towards the positively charged anode, travelling through the polymer
that fills the capillary and acts as the sieving matrix. Urea and 2-pyrrolidinone
in the gel polymer and a temperature of 60
◦
C help to prevent the formation of
any secondary structure during electrophoresis [38]. Throughout the period of elec-
trophoresis, an argon ion laser is shone through a small glass window in the capillary,
and as PCR products labelled with fluorescent dyes travel past the window they
are excited by the laser and emit fluorescence, which is detected by a charged
coupled device (CCD) camera, and then are recorded by collection software [39]
(Figure 6.4). The electrophoresis of a sample takes up to 30 minutes after which the
µ
Capillary
-ve
CCD camera
Laser
Filter
+ve
Figure 6.4
During electrophoresis an argon laser is shone through the window in the capillary. As
the labelled PCR products migrate through the gel towards the anode they are separated based on
their size. When the laser hits the fluorescent label on the PCR products it is excited and emits
fluorescent light which passes though a filter to remove any background noise and then onto a
charged coupled device camera that detects the wavelength of the light and sends the information
to a computer where software records the profile
