DETECTION OF STR POLYMORPHISMS

12 Apr

DETECTION OF STR POLYMORPHISMS

capillary electrophoresis systems and all these have multicolour detection capacity. The ABI PRISM® 310 Genetic Analyzer that has a single capillary and analyses up to 48 samples per day, the ABI PRISM® 3100 and Applied Biosystems 3130xl Genetic Analyzers, which have 16 capillaries and can analyse over 1000 samples per day, and the ABI PRISM® 3700 and Applied Biosystems 3730xl Genetic Analyzers, which can have up to 96 capillaries that can analyse over 4000 samples per day. Before electrophoresis, the PCR sample is prepared by mixing approximately 1 μl 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, heating the sam- ples to 95◦C is routinely done to ensure that the PCR products are single stranded. The samples are transferred into the capillary using electrokinetic injection, a voltage is ap- plied and charged molecules, including the amplified DNA fragments and the internal- lane size standards, migrate into the capillary. After injection, a constant voltage is ap- plied across the capillary and the PCR products migrate towards the positively charged anode, travelling through the polymer, which 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 [37]. Through- out the period of electrophoresis, 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, emit fluorescence that is detected by a charged coupleddevicecamera(CCD),andthenarerecordedbycollectionsoftware[38](Figure 6.4). The electrophoresis of a sample takes up to 30 minutes after which the polymer in the capillary is replaced with fresh polymer and the next sample can be analysed.

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, the lable is excited and emits fluorescent light that passes though a filter to remove any background noise, and then on to a charged coupled device camera that detects the wavelength of the light and sends the information to a computer where software records the profile (see plate section for full-colour version of this figure)

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