Abi Prism 3100 Genetic Analyzer 16capillary System

The multi-capillary ABI PRISM 3100 Genetic Analyzer became available in 2001 and offers a nice solution to higher throughputs with a very similar feel to the single capillary ABI 310 instrument. Both 96-well and 384-well plates of samples may be processed in the ABI 3100. With each run taking roughly 45-60 minutes, a 96-well plate can be analyzed in approximately 5-6 hours with six injections containing 16 samples each.

Several features of the ABI 3100 versus the ABI 310 are compared in Table 14.1. For example, plate records rather than sample sheets are used to designate sample positions with their names prior to collecting the STR data. Injections occur onto all 16 capillaries at once so if a user does not have 16 samples (two columns of eight samples in 96-well plate) then formamide should be put in the wells so as to not inject 'dry' which is not good for the capillaries. A photo of a capillary array as it appears inside the ABI 3100 instrument is shown in Figure 14.4.

ABI 310

ABI 3100

Number of capillaries

Cost to replace capillary or array

Laser power

Temperature control

Computer

Data storage

Sample information setup

Spectral calibration (multi-componenting matrix)

30-70°C with hot plate

Macintosh; Windows NT or 2000

Individual files in Run Folders

Sample Sheet

Can be applied to samples at any time

Number of dyes available 4- or 5-dye chemistry

Software to process STR data

GeneScan/Genotyper OR GeneMapper/D

$625 25 mW

30-70°C with enclosed oven Windows NT or 2000

Oracle database

Plate Record

Must be applied prior to sample being run

4- or 5-dye chemistry

GeneScan/Genotyper OR GeneMapper/D

Table 14.1

Comparison of several features on ABI Prism 310 and ABI Prism 3100 Genetic Analyzers.

3100 Genetic Analyzer

Figure 14.4

Photo of a 3100 capillary array illustrating the 16 glass capillaries (top) and the electrodes surrounding the capillaries (bottom) that enable electrokinetic injection of DNA samples from two columns of an 8 X 12 96-well microtiter plate.

Figure 14.4

Photo of a 3100 capillary array illustrating the 16 glass capillaries (top) and the electrodes surrounding the capillaries (bottom) that enable electrokinetic injection of DNA samples from two columns of an 8 X 12 96-well microtiter plate.

Figure 14.5 Comparison of matrix samples used for spectral calibration in (a) ABI 310 and (b) ABI 3100 instruments.

ABI 310 Matrix Samples

(b) ABI 3100 Matrix (Spectral Calibration) Sample

Blue (5FAM)

J_J_JJLLL

11

Green (JOE)

J_J_JJLU_

11

Yellow (NED)

Red (ROX)

J_J_LUlL

A 1

Separate samples run for each dye color Each sample contains multiple peaks All peaks labeled with the same dye color

Single sample run containing all dye colors

Only one peak per dye color

Injected into each capillary of the array

A separate spectral calibration file is created for each capillary

An important difference between the ABI 310 and 3100 is that spatial and spectral calibrations are required prior to collecting data on the ABI 3100. The spatial calibration enables the CCD detector to know the location of each capillary while spectral calibration is essentially the same as a matrix file generated with ABI 310 runs (see Figure 13.6). A comparison of the format for the matrix standards used to enable dye color deconvolution is illustrated in Figure 14.5. With the ABI 310 separate samples are run representing each dye color that contain multiple peaks and the same dye color. The ABI 3100 on the other hand uses a single sample within each capillary that contains one peak per dye color (Figure 14.5). The spectral calibration (color deconvolution matrix) is applied during data collection on the ABI 3100 meaning that a sample must be rerun if a different matrix is desired for a particular sample.

Newer versions of ABI 3100 data collection software (versions 1.1, 2.0, and above) tweak the red and green color channels to balance signal with 5-dye chemistry kits such as Identifiler. Data files are also stored within an Oracle database on the ABI 3100 rather than just individual files such as on the ABI 310.

Precision studies conducted on the ABI 3100 (Sgueglia et al. 2003, Butler et al. 2004) and the ABI 3700 (Gill et al. 2001) have demonstrated that reliable results can be obtained with a multi-capillary CE system.

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