The ABI Prism 310 Genetic Analyzer

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Presentation transcript:

The ABI Prism 310 Genetic Analyzer Chapter 11 The ABI Prism 310 Genetic Analyzer ©2002 Academic Press

ABI Prism 310 Genetic Analyzer capillary Syringe with polymer solution Autosampler tray Outlet buffer Injection electrode Inlet buffer ©2002 Academic Press

Close-up of ABI Prism 310 Sample Loading Area Autosampler Tray Sample Vials Electrode Capillary ©2002 Academic Press

Sample Processing Steps with ABI 310 Replace capillary Refill syringe with polymer solution Fill buffer vials Performed only once per batch of ~96 samples Prepare samples (denature, cool, and mix with size standard) Prepare sample sheet and injection list Allelic ladder every tenth injection ©2002 Academic Press

Sample Processing Steps (cont.) Automated Sample Injection, Electrophoresis and Data Collection Size DNA Fragments GeneScan Software Genotype STR alleles Genotyper Software Perform Data Analysis Manually inspect the data ELECTROPHORESIS and DETECTION steps are simultaneous ©2002 Academic Press

Quality of Formamide Affects Sensitivity ©2002 Academic Press Figure courtesy of Bruce McCord, Ohio University

Steps Performed in Standard Module Capillary fill – polymer solution is forced into the capillary by applying a force to the syringe Pre-electrophoresis – the separation voltage is raised to 10,000 volts and run for 5 minutes; Water wash of capillary – capillary is dipped several times in deionized water to remove buffer salts that would interfere with the injection process Sample injection – the autosampler moves to position A1 (or the next sample in the sample set) and is moved up onto the capillary to perform the injection; a voltage is applied to the sample and a few nanoliters of sample are pulled onto the end of the capillary; the default injection is 15 kV (kilovolts) for 5 seconds Water wash of capillary – capillary is dipped several times in waste water to remove any contaminating solution adhering to the outside of the capillary Water dip – capillary is dipped in clean water (position 2) several times Electrophoresis – autosampler moves to inlet buffer vial (position 1) and separation voltage is applied across the capillary; the injected DNA molecules begin separating through the POP-4 polymer solution Detection – data collection begins; raw data is collected with no spectral deconvolution of the different dye colors; the matrix is applied during Genescan analysis ©2002 Academic Press