Homogeneous Polymerase Chain Reaction Nucleobase Quenching Assay to Detect the 1-kbp Deletion in CLN3 That Causes Batten Disease Paul G. Rothberg, Denia.

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Presentation transcript:

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, Sharon D. Frazier, David A. Pearce The Journal of Molecular Diagnostics Volume 6, Issue 3, Pages 260-263 (August 2004) DOI: 10.1016/S1525-1578(10)60519-3 Copyright © 2004 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Strategy. A: The positions of the primers (arrows) and probe on the wild-type and mutant CLN3 gene are shown. The fluorophore on the probe is indicated with an asterisk; not drawn to scale. B: The sequence of the probe is shown in between the complementary sequences of the wild-type and deletion alleles, with vertical lines connecting the base-paired residues. The –F on the 3′ end of the probe indicates the 6-FAM fluorophore and shows its position with respect to the G residues on the opposite strand. The probe is fully base-paired with the mutant sequence, but has three unmatched nucleotides at the 5′ end when annealed to a wild-type amplicon. The G residues that contribute to the quenching of the fluorescent signal are underlined in the normal and mutant sequences. The Journal of Molecular Diagnostics 2004 6, 260-263DOI: (10.1016/S1525-1578(10)60519-3) Copyright © 2004 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Graph of -d(F1)/dT versus temperature showing the melting troughs that reveal the genotype. The direction of change in fluorescence intensity when the quenched probe dissociates from the amplicon (increase) is the opposite of that seen when using a dual probe fluorescence resonance energy transfer system to generate the fluorescent signal.14 For this reason we see a trough, or inverted peak, when using the software on the Roche LightCycler. The figure shows results for two template DNAs that are homozygous wild-type, two that are homozygous mutant, two heterozygotes, and one no template control. The Journal of Molecular Diagnostics 2004 6, 260-263DOI: (10.1016/S1525-1578(10)60519-3) Copyright © 2004 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions