All samples were diagnosed as colon or rectal cancer by hematoxylin and eosin stain. All tissues were primary CRC tissue surgically removed prior to other clinical treatments. Tissues were sliced to an approximate size of 1.0 cm2 �� 10 ��m by microtome. The sliced section samples used for this citation study were not performed by manual microdissection (MMD). No further information including demographic and clinical data were requested for these samples. The study has been reviewed and approved by the Institutional Review Board of the University of Chicago. AMDS AMDS is a fully automated genetic analytical system based on a DNA-chip which has 23 reaction wells containing reagents for PCR and Invader? assays (Figure 1A and 1B).
When a user adds a sample (whole blood, purified DNA or tissue homogenate) to the DNA purification cartridge and starts the attached software, AMDS performs DNA extraction, transfers the DNA to the chip, performs PCR and the Invader? assay, reads the results, and displays judging result in about 70 minutes. Assay flow of mutation detection is shown in Figure 1C. In step 1, DNA is extracted and purified by the DNA purification cartridge; in step 2, the purified DNA fluid sample is transferred to the DNA-Chip; in step 3, InvaderPlus? (PCR and Invader? reaction continuously in the same tube) is conducted; in the last step, AMDS reports a genotyping result of the sample. InvaderPlus? was performed under the following conditions: denature for 2 min at 93��C, followed by 30 or 35 cycles of 31 seconds at 93��C and 16 seconds at 66��C, and Taq polymerase deactivation for 2 minutes at 97��C, followed by 10 minutes of signal detection at 61��C.
Fluorescence signal of FAM (Fluorescence aminohexyl) was monitored in channel F1 at 520 nm with excitation of 490 nm, and fluorescence signal of RED (Redmond Red) was monitored in channel F2 at 595 nm with excitation of 580 nm. Figure 1 AMDS mutation detection system. DNA-chip and DNA Purification Cartridge All DNA chips and DNA purification cartridges were manufactured in a clean room at the level of ISO class 8. Required reagents mixture (1.99 ��l) for a DNA chip containing 0.1 ��l of 1 M MOPS buffer (pH 7.7) (DOJINDO LABORATORIES, Kumamoto, Japan), 0.05 ��l of 10 mM each deoxyribonucleoside triphosphate (Roche, CA, USA), 0.96 ��l of 1 M trehalose (Hayashibara, Okayama, Japan) aqueous solution, 0.
60 ��l of 20 �� oligo mix, 0.22 ��l of 5.0 U/��l Hawk Taq polymerase (Roche, CA, USA), 0.04 ��l of 15,000 U/��l Cleavase (Hologic, WI, USA) were dispensed and dried Drug_discovery in wells of a DNA chip. 20 �� oligo mixture was prepared with 0.06 ��l of 100 ��M forward primer, 0.06 ��l of 100 ��M reverse primer, 0.06 ��l of 50 ��M of FAM-FRET (Fluorescence resonance energy transfer) cassette (Hologic, WI, USA), 0.