John M. S. Bartlett.pdf - Bio-Nica.info

304 Edwards and Bartlett
11. Hot start Taq polymerase, 5 units/µL, reaction concentration is 0.5 units/reaction (Qiagen
Ltd, UK).
12. 10× reaction buffer and 25 mM MgCl 2 (Qiagen Ltd, UK); reaction concentration is
1× reaction buffer and 3 mM MgCl 2 .
13. dNTPs (10 mM; Advanced Biotech); reaction concentration is 200 µm of each dNTP.
14. PCR-grade water (Sigma Aldrich, UK).
15. Formamide (BDH, UK).
16. Loading buffer (50 mg/mL blue dextran, 25 mM EDTA).
17. Genescan 400 HD size standard (rox), (ABI Perkin–Elmer, UK).
18. Acrylamide (Sigma Aldrich, UK).
19. Bisacrylamide (Sigma Aldrich, UK).
20. Automated laser-activated fluorescent DNA sequencer, ABI 377 sequencer (Perkin
Elmer, UK).
21. Genescan and Genotyping software (Perkin Elmer, UK).
3. Methods
3.1. Microdissection of Archival DNA
1. Archival formalin-fixed, paraffin-embedded TCCs are cut into 5-µm thick sections and
put on to glass slides.
2. Tissue sections are dewaxed by incubating in xylene (2 × 10 min).
3. Tissue is rehydrated by incubating in a series of alcohol solutions, 100% alcohol
(2 × 2 min), 95% alcohol (2 min), and 70% alcohol (2 min).
4. Sections are stained in 0.05% Toluidine blue for 30 s.
5. Areas of tumor cell are microdissected from 5-µm sections using a dissecting microscope
and a micromanipulator.
6. Dissected tissue is placed in an RNA/DNA free tube containing 12 µL of protein digestion
buffer.
7. DNA is extracted by addition of a 13 µL of protein digestion buffer containing proteinase
K and incubating for 4 to 7 d at 37°C in a water (9).
8. On removal from the water bath, proteinase K is inactivated by heating for 10 min at 95°C
using a thermal cycler (see Note 1).
9. Normal tissue is also dissected from the same section as the tumor, thus providing normal
DNA to be used as a control in MIS and LOH analysis.
10. LOH and MIS analysis is conducted using 1-µL aliquots of DNA without further
purification.
3.2. Primers and Loci Analyzed
Primer sequences used for amplification of 9 microsatellites are found in Table 1.
Two microsatellites (D9S126 and D9S259) are located on the short arm of chromosome
9 spanning the 9p21 region, five microsatellites (D9S275, D9S195, D9S258, D9S103,
and GSN) are located on the long arm of chromosome 9 spanning the 9q 32-33 region
and two microsatellites (D9S1199 and D9S1198) are located on the long arm spanning
the 9q 34 region. Primers were purchased from MWG-Biotech UK Ltd, one primer
from each pair was fluorescently labeled at the 5′ end.
3.3. PCR
The target sequences were amplified by PCR in 10-µL reactions, with each reaction
containing the following.