Chapter 11

The ABI Prism 310 Genetic Analyzer

©2002 Academic Press

capillary

Syringe with polymer solution

Autosampler tray

Outlet buffer

Injection electrode

Inlet buffer

ABI Prism 310 Genetic Analyzer

©2002 Academic Press

Autosampler Tray

Sample Vials

ElectrodeCapillary

Close-up of ABI Prism 310 Sample Loading Area

©2002 Academic Press

Sample Processing Steps with ABI 310

   

Prepare samples(denature, cool, and mix with size

standard)

Replace capillary

Refill syringe with polymer solution

Fill buffer vials

Performed only once per batch of ~96 samples

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

208 S

1180 S

408 S

338 S

Figure courtesy of Bruce McCord, Ohio University©2002 Academic Press

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