A subscription to JoVE is required to view this content. Sign in or start your free trial.
Chip-in-a-Tube-Based Digital PCR for Quantification of Single Nucleotide Variants
Overview
This video demonstrates the utility of digital PCR in a chip-in-a-tube format for the detection of single nucleotide variations in DNA samples. A single PCR reaction is partitioned into chambers that act as independent PCR reactions, and the detection of fluorescence signals from the amplified targets in the chambers is used to compute the frequency of the variant allele in the sample.
Protocol
1. Quality Control of Genomic DNA
NOTE: Genomic DNA (gDNA) was extracted from peripheral blood using the well-established silica-membrane-based DNA purification method. In advance of the below procedures, the concentration of gDNA was determined using a spectrophotometer.
- Prepare the gDNA sample at a concentration of 10-100 ng/µL.
- Add 10 µL of DNA sample buffer to an 8-tube strip.
- Add 1 µL of DNA ladder or gDNA to the tubes. .......
Representative Results
Figure 1: Points to note for the dPCR assay with chip-in-a-tube format. (A) This panel shows how to set up the 8-tube strip in the autoloader. (B) This panel shows broken chips. (C) This panel shows how the chip surfaces just after (i) loading and (ii) sealing. (iii) A puddle of liquid is visible in the case of an incomplete sealing. (
This article has been published
Video Coming Soon
Source: Kahyo, T., et al. Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format. J. Vis. Exp. (2018).
ABOUT JoVE
Copyright © 2025 MyJoVE Corporation. All rights reserved