 |
Department of Clinical Oncology
|
 |
|
 |
|
|
|
|
|
|
|
|
|
|
|
Protocol for Chromosome Microdissection Here we are not focusing on chromosome harvest, please refer to the CGH protocol for further information.
Fig.1 Main procedure of Chromosome Microdissection
FIRST STEP: MICRODISSECTING Materials: Metaphase (Store in fixative in-20°C), Fixative (Methanol : glacial acetic acid is 3:1, fresh prepared) 1.2-mm capillaries (World Precision Instruments, Sarasata FL, 1B1 20F4) 24×60mm coverslips, 2% Trypsin (Lifetechnologies) Giemsa stain (Lifetechnologies, Grandisland, NY, 10092-013) Topoisomerase I (Promega Corp., Madison, WI, M2851) 5×Topo buffer (200mmol/L Tris-HCl (pH7.5), 100mmol/L MgCl2, 250mmol/L NaCl) 10×dNTP (0.2mM) (100mM dNTP(Pharmacia LKB, Piscataway, NJ, 27-2045-01) diluted by water) DOP-PCR primer (UN1: 5’-CCGACTCGAGNNNNNNATGTGG-3’),diluted to 100uM
Procedure: 1. Warm metaphase to room temperature; 2. Centrifuge at 1000rpm for 5min, discard the supernatant; 3. Wash the palette twice with fresh made fixative, 4. Drop the metaphase on the coverslip, airdry. 5. Incubate at 37°C for 3 days. 6. Trypsinize the metaphase and then stain at 37.5% Giemsa stain for 5 minutes. 7. Air dry. Incubate at 37°C overnight. 8. Glass needles are prepared on a automatic pipette puller(Narishige, Japan, PC-10) 9. Mix the following to prepare collection buffer
Aliquots the collection buffer to 4 PCR tubes, 5ul each 10. Place the coverslip on a inverted microscope, the glass needle is controlled by a hydraulic micromanipulator (Narishige, Japan, MO-302) 11. Under the microscope, dissect the region which you are interested in, and casually transfer it into the collection buffer, place on ice. 12. Dissect 4~8 copies of interested DNA, then incubate the PCR tubes on PCR machine at 37°C for 1 hour.
SECOND STEP: PROBE AMPLIFICATION AND LABELING Materials: T7 Sequenase (US Biochemicals, Cleveland, OH, 70702) Enzyme dilution buffer (obtain from the T7 sequenase kit, aliquots to 7ul per tube, store at -20°C) 10×dNTP (0.2mM) (100mM dNTP(Pharmacia LKB, Piscataway, NJ, 27-2045-01) diluted by water) DOP-PCR primer (UN1: 5’-CCGACTCGAGNNNNNNATGTGG-3’),diluted to 100uM AmpliTaq® DNA Polymerase, LD (Perkin-Elmer, Foster City, CA, N808-0157) 10×PCR reaction buffer (100mmol/L Tris-HCl (pH 8.4), 40mmol/L MgCl2, 500mM KCl, 0.01%Gelatin) Biotin-16-dUTP or Spectrum Orange or other fluorescence dye (1mM). 3M sodium acetate (pH5.2) cold absolute ethanol
Procedure: 1. Sequenase is diluted eight-fold by add 1ul sequenase to 7ul enzyme dilution buffer. 2. Synthesis of primer-terminated copies. This is achieved by 6~8 cycles of denaturation, primer annealing, and low temperature primer extension using a highly processive polymerase-Sequenase. The dissected products are heated to 94°C for 5min, then In each priming cycle, the temperature are 94°C for 1min, 30°C for 3min, and 37°C for 2min. A 0.2ul portion of diluted sequenase is added to the tube at each period of 30°C incubation. 3. The primer-terminated DNA strands are then amplified in a PCR reaction. Mix the following according to the recipe here:
Add the mixture to the tube. The PCR cycle is: 94°C for 5min, then followed by 30 cycles of 94°C 1min, 56°C 1min, and 72°C 1.5min. The products are finally extended at 72°C for 5min. Thus the first-round of PCR is finished. 4. The first-round PCR products are amplified in another PCR reaction which is done by adding 2ul first-round PCR product to the similar system of first PCR. The second PCR is 25 cycles. And the temperature condition is similar to those of first PCR. 5. Test PCR efficiency by agarose gel electrophoresis. 6. If the PCR is good, label the PCR products in another PCR reaction that contain fluorescence as follows:
7. Add one-tenth volume of 3M sodium acetate (pH5.2) into the PCR products, mix well, then add two-fold volume of cold absolute ethanol into it, mix well. 8. Incubate for 2hr at -20°C. 9. Use FISH test your chromosome microdissection. (See Protocol for FISH)
|
|
|