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

Abstract

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.

Standardized assessment of behavior and adaptive living skills in juvenile neuronal ceroid lipofuscinosis

“…Thirteen children were homozygous for the common deletion in the CLN3 gene. The common deletional mutation in the CLN3 gene was analyzed by using methodology previously described by this research group (Rothberg et al 2004). Two children were heterozygous for the deletion with a novel mutation.…”

Quantifying physical decline in juvenile neuronal ceroid lipofuscinosis (Batten disease)

“…Analysis of the common deletion in the CLN3 gene was done as previously described. 12 If the subject was not homozygous for the common deletion, additional sequencing of the CLN3 gene was performed.…”

Homogeneous PCR nucleobase quenching assays to detect four mutations that cause neuronal ceroid lipofuscinosis: T75P and R151X in CLN1, and IVS5-1G>C and R208X in CLN2

“…We attempted to design assays using a homogeneous PCR platform that uses differential melting of an oligodeoxyribonucleotide probe to distinguish the alleles, and nucleobase quenching of a fluorescent dye linked to the probe to generate the signal, because we have found this method useful for low-volume genotyping in several situations including detection of the most common mutation in the CLN3 gene (Rothberg et al, 2004).…”

“…We made use of a strategy that employs PCR, differential melting of a fluorescently labeled oligodeoxyribonucleotide probe to distinguish the alleles, and nucleobase quenching to detect probe hybridization (Crockett and Wittwer, 2001). We have found that this strategy is useful for low-volume genotyping for rare diseases (Rothberg et al, 2004;McClaskey et al, 2005).…”