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Methods UsefuJ in Genetics and OncoJogy
such as the common ~F508- and the M507 -mutations, in fragments up to 90bp by
heteroduplex formation. A distinction of the ~F508- and ~1507-homoduplices
from wildtype-homoduplices was possible in fragments up to a length of 76bp.
In addition, we tested the Ibp-insertion 3905insT and the point mutation
G542X (G~T) in small fragments «80bp). We did not detect a significant difference for the 3905insT in a hetero- and a homozygote sample. For the G542X a heterozygote sample was analyzed and a small shift of T m was detected.
In conclusion, we showed that the melting curve analysis of the LightCycler
System using the SYBR Green I dye can be used for fragment differentiation and
is even capable to detect small mutations in small fragments.
Materials
Equipment LightCycler Instrument (Roche Diagnostics, Switzerland)
Oligo primer analysis software 6 for Macintosh (MedProbe, Norway)
Reagents Oligonucleotides (Microsynth, Switzerland)
LightCycler-DNA Master SYBR Green I (Roche Diagnostics)
DNA extraction spin columns (Qiagen, Switzerland)
Procedure
Sample preparation Human total DNA was extracted from peripheral blood cells via DNA extraction
spin columns (Qiagen, Switzerland). DNA was used at a concentration of 25 ng/Ill.
Primer design Primers (Table 1) were designed and tested for primer dimers using the oligo primer
analysis software (MedProbe, Norway). Amplification sequences with different GC
content were designed to carry equal GC distributions. Primers for mutation detection were selected to carry the mutation in the middle of the amplification fragment.
Table 1. Oligonucleotides
CFTR Accession No.: M55107. M55115. M55116. M55127, M55129
CFTRexon
length
GC(%)
T m (0C)
Primers
AGTTTTCCTGGATTATGCCT
20
40
58.0
CTTTGATGACGCTTCTGTAT
20
40
57.0
Product I
10
90
36
76.8
Primers
GAATTTCATTCTGTTCTCAGT
21
33
55.2
CTTACCTCTTCTAGTTGGCA
20
45
57.7
Product 2
10
130
37
79
Methods UsefuJ in Genetics and OncoJogy
such as the common ~F508- and the M507 -mutations, in fragments up to 90bp by
heteroduplex formation. A distinction of the ~F508- and ~1507-homoduplices
from wildtype-homoduplices was possible in fragments up to a length of 76bp.
In addition, we tested the Ibp-insertion 3905insT and the point mutation
G542X (G~T) in small fragments «80bp). We did not detect a significant difference for the 3905insT in a hetero- and a homozygote sample. For the G542X a heterozygote sample was analyzed and a small shift of T m was detected.
In conclusion, we showed that the melting curve analysis of the LightCycler
System using the SYBR Green I dye can be used for fragment differentiation and
is even capable to detect small mutations in small fragments.
Materials
Equipment LightCycler Instrument (Roche Diagnostics, Switzerland)
Oligo primer analysis software 6 for Macintosh (MedProbe, Norway)
Reagents Oligonucleotides (Microsynth, Switzerland)
LightCycler-DNA Master SYBR Green I (Roche Diagnostics)
DNA extraction spin columns (Qiagen, Switzerland)
Procedure
Sample preparation Human total DNA was extracted from peripheral blood cells via DNA extraction
spin columns (Qiagen, Switzerland). DNA was used at a concentration of 25 ng/Ill.
Primer design Primers (Table 1) were designed and tested for primer dimers using the oligo primer
analysis software (MedProbe, Norway). Amplification sequences with different GC
content were designed to carry equal GC distributions. Primers for mutation detection were selected to carry the mutation in the middle of the amplification fragment.
Table 1. Oligonucleotides
CFTR Accession No.: M55107. M55115. M55116. M55127, M55129
CFTRexon
length
GC(%)
T m (0C)
Primers
AGTTTTCCTGGATTATGCCT
20
40
58.0
CTTTGATGACGCTTCTGTAT
20
40
57.0
Product I
10
90
36
76.8
Primers
GAATTTCATTCTGTTCTCAGT
21
33
55.2
CTTACCTCTTCTAGTTGGCA
20
45
57.7
Product 2
10
130
37
79