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Applications in Genetics
In the first month oflife, CF babies show increased concentrations of immunoreactive trypsinogen (IRT). IRT is measured from dried blood spot samples (Guthrie
cards) collected from babies 3 days after birth. However, the specificity of this test
in the first week of life is very low. Therefore, blood spots exhibiting elevated IRT
are tested for common CF mutations. This so-called two-tiered IRT/DNA strategy
has been shown to result in a good positive predictive value for CF [7,8].
Here, we describe a procedure to rapidly detect five common CF mutations
from dried blood spots using fluorogenic hybridization probes and dual color
melting point analysis.
Materials
Equipment LightCyder instrument
Reagents QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany)
Proteinase K (Qiagen)
Sample Preparation
Primer and Probe
Design
Amplification Primer (TIB MOLBIOL, Berlin, Germany; GENSET, Paris, France)
Hybridization Probes (TIB MOLBIOL; GENSET)
LightCyder FastStart DNA Master Hybridization Probes (Roche Diagnostics,
Mannheim, Germany)
DNAs with different CF mutations were kindly provided by T. Meitinger (Technische Universitat Miinchen, Germany)
Procedure
For DNA extraction, the QIAamp DNA Blood Mini Kit (Qiagen) was used. Two
spots with a diameter of 3 mm were punched out from dried blood spots on
Guthrie fllter paper cards. In total, 180 j.il of buffer ATL (Qiagen) was added to the
spots. After incubation for 10 min at 85°C, 25 j.il of Proteinase K (>600 mAU/ml)
was added, followed by incubation for 60 min at 56°C. The remaining steps in
DNA purification were performed according to the manufacturer's instructions.
DNA was eluted in 50 fll5 mmo1!1 Tris (pH 7.5).
Since the test is planned to be used in a CF newborn screening program in Bavaria,
Germany, the five most frequent Bavarian CF mutations, i.e., M508, R347P, G542X,
G55ID, and R553X were chosen [5]. The oligonudeotides used for the detection of
different CF alleles are shown in Table 1. Two primer pairs are necessary to amplify the R347P and the M50810ci in the CFTR gene. The amplification results in PCR
products of 187 bp and 218 bp, respectively. The hybridization probe systems for
melting point analysis of the two loci are labelled with different dyes, i.e., LCRed640
and LCRed705, to allow multiplex detection in a single reaction. The R347P detection probe contains a mismatch to avoid probe dimerization. A single primer pair
is designed to amplify a 240-bp CFTR gene region encompassing the G542X,
G551D, and R553X mutations. Two hybridization probe systems, again labelled with
Applications in Genetics
In the first month oflife, CF babies show increased concentrations of immunoreactive trypsinogen (IRT). IRT is measured from dried blood spot samples (Guthrie
cards) collected from babies 3 days after birth. However, the specificity of this test
in the first week of life is very low. Therefore, blood spots exhibiting elevated IRT
are tested for common CF mutations. This so-called two-tiered IRT/DNA strategy
has been shown to result in a good positive predictive value for CF [7,8].
Here, we describe a procedure to rapidly detect five common CF mutations
from dried blood spots using fluorogenic hybridization probes and dual color
melting point analysis.
Materials
Equipment LightCyder instrument
Reagents QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany)
Proteinase K (Qiagen)
Sample Preparation
Primer and Probe
Design
Amplification Primer (TIB MOLBIOL, Berlin, Germany; GENSET, Paris, France)
Hybridization Probes (TIB MOLBIOL; GENSET)
LightCyder FastStart DNA Master Hybridization Probes (Roche Diagnostics,
Mannheim, Germany)
DNAs with different CF mutations were kindly provided by T. Meitinger (Technische Universitat Miinchen, Germany)
Procedure
For DNA extraction, the QIAamp DNA Blood Mini Kit (Qiagen) was used. Two
spots with a diameter of 3 mm were punched out from dried blood spots on
Guthrie fllter paper cards. In total, 180 j.il of buffer ATL (Qiagen) was added to the
spots. After incubation for 10 min at 85°C, 25 j.il of Proteinase K (>600 mAU/ml)
was added, followed by incubation for 60 min at 56°C. The remaining steps in
DNA purification were performed according to the manufacturer's instructions.
DNA was eluted in 50 fll5 mmo1!1 Tris (pH 7.5).
Since the test is planned to be used in a CF newborn screening program in Bavaria,
Germany, the five most frequent Bavarian CF mutations, i.e., M508, R347P, G542X,
G55ID, and R553X were chosen [5]. The oligonudeotides used for the detection of
different CF alleles are shown in Table 1. Two primer pairs are necessary to amplify the R347P and the M50810ci in the CFTR gene. The amplification results in PCR
products of 187 bp and 218 bp, respectively. The hybridization probe systems for
melting point analysis of the two loci are labelled with different dyes, i.e., LCRed640
and LCRed705, to allow multiplex detection in a single reaction. The R347P detection probe contains a mismatch to avoid probe dimerization. A single primer pair
is designed to amplify a 240-bp CFTR gene region encompassing the G542X,
G551D, and R553X mutations. Two hybridization probe systems, again labelled with