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DNA extraction with the Magtration System
The detailed procedure is the same as that specified in the manual for commercially available devices of
this kind. In the present experiment, genomic DNA purified in advance (20 ng/sample) was used.

PCR & Labeling
The PCR mixture is composed of distilled water (12.5 ul), 10-fold buffer (2.0 ul),50mM MgCl2 (0.8ul), 0.5
mM dATP, dCTP, dGTP each, 0.475 mM dTTP, 25 uM digoxigenin-11-dUTP, 4SNP Primer Mix (1.6 ul),
HOTGoldstar™ DNA Polymerase (Eurogentec, 0.1 ul) and DNA sample (1 ul). PCR was performed using a
Dice® Gradient (Takara Bio) for amplification and elongation through one cycle at 95°C for 10 minutes,
33 cycles at 95°C for 30 seconds, 60°C for 30 seconds and 72°C for 30 seconds and one cycle at
95°C for 5 minutes. .

Luminescence detection with BIST
The hybridization buffer, one of the reagents for BIST, is combined with 5.0 ul of the PCR amplicon. This is
followed by hybridization with BIST and the Magtration System. The chemiluminescence reagent (
Luminol/Enhancer Solution) and the Stable Peroxide Solution of SuperSignal® West Femto Maximum
Sensitivity Substrate (PIERCE) are combined in a ratio of 1:1, to yield the substrate.
The BIST capillary is mounted on the end of a pipette and the substrate is aspirated. Then, the sample is set
on the BISTnner™ and scanned. The intensity of luminescence (signal) on the bead surface is detected, and
the results are compared with the information as to the locations of beads

BIST Multiplex DNA typing

First, a primer designed for the specific SNP sequence for each allele is synthesized. When designing the primer,
cmpTAG oligonucleotide, complementary to the TAG oligonucleotide fixed to the BIST beads are added to the 5’
-terminal end. If the SNP type is A or G , a yellow fluorescence indicates T and pink indicates C. In type AA
individuals, only the complementary primer undergoes elongation . Because some of the dUTP has been labeled,
the elongation product hybridized with the BIST beads has incorporated the label. The primer without a
complementary terminal does not undergo elongation because it does not match the site of the SNP. In this way,
only the primer is hybridized to the BIST beads. The elongation product obtained by this mechanism contains the
label. If the label is exposed to the antibody contained in the POD-adsorbed label, the POD used for detection of
chemiluminescence is labeled. Subsequent exposure to the substrate for POD allows detection of light from the
beads that are bound to the elongated primer. Each of these processes has been automated, using the PSS’s
Magtration System and BIST Scanner, utilizing a commercially available thermal cycler.

Sample C

－Result of Sequence by ABI sequencer－

The BIST capillary was mounted on a hand held pipette and the substrate was aspirated and the sample was
placed in the BISTnner™. The display screen, presenting the results of scanning. For each polymorphism, a
single bar graph was depicted in cases of homozygotes (Homo), while double bar graphs were formed for
heterozygotes (Hetero), thus allowing diagnosis in an easily discernible manner. Confirmation of the results
from the BIST system were in 100 % agreement with sequence results obtained by ABI sequencer.

Sample B

Sample A

Results

Materials and Methods