Rapid Screening for Five Major Cystic Fibrosis Mutations by Melting Peak Analysis •
Results
Figure 1 shows the results of a CF genotyping experiment with four different DNA
samples.
Agarose gel electrophoresis of products of llF5081R347P multiplex PCR results
in two bands of 187 bp and 218 bp (Fig. la). Fluorescence monitoring of the
hybridization probes for R347P detection is shown in Fig. lb. Samples 3-5 exhibit one wild-type peak at 62°C. Sample 2 is heterozygous for R347P with the wildtype peak at 62°C and a mutation peak at 51°C. Figure 1 c shows the melting point
analysis of the llF508 deletion locus. Samples 2 and 3 are heterozygous for the
deletion (peaks at 66°C and 57°C, respectively). The other samples exhibit only the
wild-type peak at 66°C (Table 2).
Only one band (240 bp) is generated with the G542XIG55IDIR553X PCR by
agarose gel electrophoresis (Fig. Id). The melting point analysis of the
G551DIR553X locus is shown in Fig. Ie. Samples 2 and 3 exhibit one wild-type
peak at 65°C. Sample 5, which is heterozygous for R553X, shows two peaks: the
wild-type peak at 65°C and a mutation peak at 59°C. Sample 4 is heterozygous for
G551D; however, due to the small T m difference between wild type and mutation,
it is difficult to obtain a separation of the two melting peaks. Only one broad peak
is visible at 62°C. From this analysis it is not possible to conclude whether this
sample is heterozygous or homozygous for the G551D mutation. Figure If shows
the detection of the G542X mutation. Sample 3 is heterozygous for this mutation,
exhibiting peaks at 63SC and 57°C for wild type and mutation, respectively. All
other samples are wild type (one peak at 63SC). The genotypes of the samples
used in this study are summarized in Table 3.
Comments
Neonatal CF screening from Guthrie card blood samples by measuring IRT shows
only a very low predictive value. Many unaffected babies also have high IRT values. Another problem is the contamination of samples with feces, resulting in
erroneously elevated IRT values. A second test is therefore necessary to confirm
CF. Genetic screening appears to be the most specific procedure. The high number of tests in a screening program requires a fast, safe, and easy-to-interpret test.
A test based on melting point analysis of fluorescently labelled probes after high
speed PCR amplification on a LightCycler instrument definitely meets these
requirements. After nucleic acid extraction from the blood spots, five major cystic fibrosis mutations can be detected in less than 1 h.
The detection of a specific mutation should be verified by a separate test using
a mutation-specific probe since there are several other, though extremely rare,
CF-causing mutations, which also can cause a T m shift in the melting analysis
using the described probes. For example, Lli507 is detected with the LlF508
hybridization probes and shows the same T m shift as a LlF508 deletion.
Running the entire test in a single capillary could be a goal for future progress
in the technique. With the present probe design the probes labelled with the same
Results
Figure 1 shows the results of a CF genotyping experiment with four different DNA
samples.
Agarose gel electrophoresis of products of llF5081R347P multiplex PCR results
in two bands of 187 bp and 218 bp (Fig. la). Fluorescence monitoring of the
hybridization probes for R347P detection is shown in Fig. lb. Samples 3-5 exhibit one wild-type peak at 62°C. Sample 2 is heterozygous for R347P with the wildtype peak at 62°C and a mutation peak at 51°C. Figure 1 c shows the melting point
analysis of the llF508 deletion locus. Samples 2 and 3 are heterozygous for the
deletion (peaks at 66°C and 57°C, respectively). The other samples exhibit only the
wild-type peak at 66°C (Table 2).
Only one band (240 bp) is generated with the G542XIG55IDIR553X PCR by
agarose gel electrophoresis (Fig. Id). The melting point analysis of the
G551DIR553X locus is shown in Fig. Ie. Samples 2 and 3 exhibit one wild-type
peak at 65°C. Sample 5, which is heterozygous for R553X, shows two peaks: the
wild-type peak at 65°C and a mutation peak at 59°C. Sample 4 is heterozygous for
G551D; however, due to the small T m difference between wild type and mutation,
it is difficult to obtain a separation of the two melting peaks. Only one broad peak
is visible at 62°C. From this analysis it is not possible to conclude whether this
sample is heterozygous or homozygous for the G551D mutation. Figure If shows
the detection of the G542X mutation. Sample 3 is heterozygous for this mutation,
exhibiting peaks at 63SC and 57°C for wild type and mutation, respectively. All
other samples are wild type (one peak at 63SC). The genotypes of the samples
used in this study are summarized in Table 3.
Comments
Neonatal CF screening from Guthrie card blood samples by measuring IRT shows
only a very low predictive value. Many unaffected babies also have high IRT values. Another problem is the contamination of samples with feces, resulting in
erroneously elevated IRT values. A second test is therefore necessary to confirm
CF. Genetic screening appears to be the most specific procedure. The high number of tests in a screening program requires a fast, safe, and easy-to-interpret test.
A test based on melting point analysis of fluorescently labelled probes after high
speed PCR amplification on a LightCycler instrument definitely meets these
requirements. After nucleic acid extraction from the blood spots, five major cystic fibrosis mutations can be detected in less than 1 h.
The detection of a specific mutation should be verified by a separate test using
a mutation-specific probe since there are several other, though extremely rare,
CF-causing mutations, which also can cause a T m shift in the melting analysis
using the described probes. For example, Lli507 is detected with the LlF508
hybridization probes and shows the same T m shift as a LlF508 deletion.
Running the entire test in a single capillary could be a goal for future progress
in the technique. With the present probe design the probes labelled with the same