Novel Chemiluminescent Substrate and Probe Systems for the Identification of CFDeltaF508 Genotypes.
Background: Chemiluminescence detection systems are rapidly gaining popularity as safer alternatives to isotopic methods in molecular diagnostics with equal sensitivity and specificity. In addition, they offer versatility of detection because of the availability of different haptens for labeling the probes, the antihapten antibodies conjugated with either alkaline phosphatase (AP) or horseradish peroxidase (HRP), and their respective chemiluminescent substrates. A novel dual chemiluminescent substrate (AP and HRP based) and probe systems to distinguish genotypes of cystic fibrosis DeltaF(508) mutation are described. Methods and Results: Two methodologies have been formulated to identify positively the genotypes of the cystic fibrosis DeltaF(508) mutation. In method 1, a pair of oligonucleotides designed to anneal to the fanking regions of DeltaF(508) mutation are differentially labeled with the hapten biotin or fluorescein and ligated using the template DNA of wild-type (N/N), heterozygous (N/DeltaF(508)), and homozygous (DeltaF(508)/DeltaF(508)) genotypes. The ligated product containing both labels is detected by first binding with avidin-HRP and anti-fluorescein-AP followed by reaction with the dual substrate. As expected, the ligation products are detected only in n/DeltaF(508) and DeltaF(508)/DeltaF(508) genotypes but not in N/N, where the ligation is precluded by the presence of three intervening nucleotides. In method 2, the three genotypes are hybridized on a membrane simultaneously with uniquely labeled (biotin or digoxigenin) oligonucleotides each designed to bind either the normal or the mutant allele. On treatment with HRP- and AP-conjugated antibodies followed by reaction with the dual substrate, only the band from N/DeltaF(508) genotype emitted a strong signal because of the binding of both oligonucleotides. Conclusions: The ligation and hybridization methods in conjunction with the dual substrate can detect and differentiate the genotypes with the DeltaF(508) mutation. These formats may be valuable for distinguishing normal individuals from carriers in population screening and fetuses that are heterozygous, from those that are homozygous for cystic fibrosis DeltaF(508) in prenatal and neonatal diagnosis.
Reddy
LV
Department of Clinical Pathology, William Beaumont Hospital, Royal Oak, Michigan, USA
Akhavan-Tafti
H
DeSILVA
R
Handley
R
Farkas
DH
Schap
AP