gg Applications in Oncology
• Melting curve analysis BAT26
Parameter
Value
Cycles
Type
Melting curve
Segment 1
Segment 2
Segment 3
Target temperature [0C]
95
35
95 (BAT26)
6S (BAI2S)
Incubation time [sl
0
30
0
Temperature transition rate lOC/s)
20
20
0.2
Acquisition mode
None
None
Continuous
Gains
Fl=l; F2=10; F3=1 (BAT26)
PI= l; F2=1; F3= 10 (BAT2S)
Results
Successful amplification and melting point analysis of BAT26 was feasible in 65 of
81 (80%) tumor samples and in 640f 81 (79%) normal mucosa samples. Melting
temperature (Tm) of DNA from normal mucosa and control DNA from mononuclear blood cells was 51.0-S1SC (Fig. la). In contrast, the Tm of DNA from all 16
tumors with MSI-H phenotype, as determined by fragment analysis using the
ABI3lO sequencer and loss of hMSH2, hMLHI or hMSH6 protein expression,
shifted to 42-S0°C (Fig. Ib). Fragment analysis using the ABI3lO capillary
sequencer revealed that the T m alterations reflect shortenings of 3-12 bases in the
repetitive stretch of 26 adenosines within the BAT26 marker.
Amplification of BAT2S was possible in 63 of 81 (78%) tumor samples and in
60 of 81 (74%) normal mucosa samples. BAT2S showed a Tm of 47-48°C in DNA
from normal mucosa, tumors without microsatellite instability, and DNA from
mononuclear blood cells (Fig. 2a). In contrast, the Tm of IS MSI-H tumors was
42.S-43.7°C (Fig. 2b), which reflects shortenings of 5-7 bases within 25 repetitive
adenines of the BAT2S marker as determined by fragment analysis.
Comments
Detection of MSI in colorectal tumors can be achieved by LightCycler melting peak
analysis using specific hybridization probes for BAT26 and BAT25. This new technique is significantly faster than conventional PCR techniques followed by electrophoretic separation of the amplicons. Furthermore, the contamination risk,
especially in a high throughput laboratory setting, is greatly reduced. Since MSI -H
is defined as the detection of at least two unstable microsatellites from a set of five
markers [S, 6], it may not be necessary to analyze more markers if MSI is detected
by BAT26 and BAT25, which are described as the most sensitive markers for MSI
detection in colorectal tumors [S, 11, 12, 13]. Most colorectal MSI-H tumors
• Melting curve analysis BAT26
Parameter
Value
Cycles
Type
Melting curve
Segment 1
Segment 2
Segment 3
Target temperature [0C]
95
35
95 (BAT26)
6S (BAI2S)
Incubation time [sl
0
30
0
Temperature transition rate lOC/s)
20
20
0.2
Acquisition mode
None
None
Continuous
Gains
Fl=l; F2=10; F3=1 (BAT26)
PI= l; F2=1; F3= 10 (BAT2S)
Results
Successful amplification and melting point analysis of BAT26 was feasible in 65 of
81 (80%) tumor samples and in 640f 81 (79%) normal mucosa samples. Melting
temperature (Tm) of DNA from normal mucosa and control DNA from mononuclear blood cells was 51.0-S1SC (Fig. la). In contrast, the Tm of DNA from all 16
tumors with MSI-H phenotype, as determined by fragment analysis using the
ABI3lO sequencer and loss of hMSH2, hMLHI or hMSH6 protein expression,
shifted to 42-S0°C (Fig. Ib). Fragment analysis using the ABI3lO capillary
sequencer revealed that the T m alterations reflect shortenings of 3-12 bases in the
repetitive stretch of 26 adenosines within the BAT26 marker.
Amplification of BAT2S was possible in 63 of 81 (78%) tumor samples and in
60 of 81 (74%) normal mucosa samples. BAT2S showed a Tm of 47-48°C in DNA
from normal mucosa, tumors without microsatellite instability, and DNA from
mononuclear blood cells (Fig. 2a). In contrast, the Tm of IS MSI-H tumors was
42.S-43.7°C (Fig. 2b), which reflects shortenings of 5-7 bases within 25 repetitive
adenines of the BAT2S marker as determined by fragment analysis.
Comments
Detection of MSI in colorectal tumors can be achieved by LightCycler melting peak
analysis using specific hybridization probes for BAT26 and BAT25. This new technique is significantly faster than conventional PCR techniques followed by electrophoretic separation of the amplicons. Furthermore, the contamination risk,
especially in a high throughput laboratory setting, is greatly reduced. Since MSI -H
is defined as the detection of at least two unstable microsatellites from a set of five
markers [S, 6], it may not be necessary to analyze more markers if MSI is detected
by BAT26 and BAT25, which are described as the most sensitive markers for MSI
detection in colorectal tumors [S, 11, 12, 13]. Most colorectal MSI-H tumors