IIIiI Application in Oncology
High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany)
LightCycler DNA Master Hybridization Probes (Roche Diagnostics)
Procedure
Sample preparation Tumor tissue and normal mucosa from 113 patients with CRC were microdissected from 5-J..Lm sections of formalin-fixed paraffin-embedded tissues by laser
assisted (P.A.L.M., cx:Germany) or manual microdissection. DNA preparation was
done using the High Pure peR Preparation Kit (Roche Diagnostics, Mannheim,
Germany) and quality of DNA was checked by electrophoresis (1.3% agarose gel).
DNA concentration was spectrophotometrically measured and 50-100 ng template DNA was used for LightCycler PCR.
Oligonucleotides Amplification of BAT26 and BAT25 markers was done with published primers
(Table 1), [10]. Hybridization probes (Table 1) were designed as far as possible
according to the guidelines recommended by the LightCycler operator's manual. The donor hybridization probes were labeled at the 3' end with fluorescein
Table 1. Oligonucleotides
BAT26 GenBank Accession #U41210
Position
Length
GC(% ) Tm (OC)
BAT26 Primers
TGACTACTTTTGACTTCAGCC
123
21
42.9
59.4
AACCATTCAACATTTTTAACCC
243R
22
31.8
57.1
Product 1
243-123
120
BAT26 Probes
GCAGCAGTCAGAGCCCTTAACCT-F
163
23
56.5
68.0
LCRed640-TCAGGTAAAAAAAAAAAAAA190
32
9.4
57.8
AAAAAAAAAAAA- P
BAT25 GenBank Accession #L04143
Position
Length
GC(% ) Tm (0C)
BAT25 Primers
TCGCCTCCAAGAATGTAAGT
6839
20
45
60.4
TCTGCATTTTAACTATGGCTC
6961R
21
38
57.5
Product 1
6739-6961 122
BAT25 Probes
CAAAAAAAAAAAAAAAAAAAAAAAAATCA-F 6925R
29
6.9
55.0
LCRed705-AACAAAACACAAAACTCTTTAGA6892R
28
28.6
60.4
GAATC-P