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JMD 2004, Vol. 6, No. 3
Copyright © 2004 American Society for Investigative Pathology & Association for Molecular Pathology

Homogeneous Polymerase Chain Reaction Nucleobase Quenching Assay to Detect the 1-kbp Deletion in CLN3 That Causes Batten Disease

Paul G. Rothberg*, Denia Ramirez-Montealegre{dagger}, Sharon D. Frazier* and David A. Pearce{dagger}{ddagger}

From the Section of Molecular Diagnosis, Department of Pathology and Laboratory Medicine, * the Center for Aging and Developmental Biology, {dagger} and the Department of Biochemistry and Biophysics, {ddagger} University of Rochester Medical Center, Rochester, New York

Batten disease is an autosomal recessive disorder also known as juvenile neuronal ceroid lipofuscinosis. The most common mutation for this disease is an approximately 1-kbp deletion in the CLN3 gene, which accounts for about 80 to 85% of the mutation load. We developed a rapid assay for this mutation using the PCR to produce amplicons that are detected by nucleobase quenching of the fluorescent signal from a probe labeled with a fluorescent dye. The probe overlaps the deletion breakpoint and is completely base paired to the mutant amplicon. However, three bases at the 5' end of the probe do not base pair with the wild-type amplicon. The alleles are distinguished by the different melting temperatures of the probe amplicon hybrids. Comparison of this new method with an allele-specific PCR and gel electrophoresis-based method showed 100% concordance in determination of the genotype for 30 specimens (11 homozygous mutant, 8 heterozygotes, and 11 homozygous normal). PCR followed by allele-specific melting curve analysis using nucleobase quenching has utility as a rapid method for detection of the most common mutation that causes Batten disease.




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