Chip-in-a-Tube-Based Digital PCR for Quantification of Single Nucleotide Variants

Abstract

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).

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. (<stron…

Materials

QIAamp DNA Blood Maxi Kit	Qiagen	51194	Before Protocol 1: Extraction of genomic DNA
NanoDrop 1000	ThermoFisher SCIENTIFIC	ND-8000	Before Protocol 1: Spectrophotometer
8-strip tube	Agilent Technologies	401428	Protocol 1
Genomic DNA sample buffer	Agilent Technologies	5067-5366	Protocol 1:
			A component of Genomic DNA Screen Tape Assay
DNA ladder	Agilent Technologies	5067-5366	Protocol 1:
			A component of Genomic DNA Screen Tape Assay
MS3 Basic Small Shaker	IKA	3617000	Protocol 1: Vortex mixer
Genomic DNA ScreenTape	Agilent Technologies	5067-5365	Protocol 1: Gel device
2200 TapeStation system	Agilent Technologies	G2965AA	Protocol 1: Electrophoresis instrument
TapeStation Analysis Software	Agilent Technologies	Bundled with G2965AA	Protocol 1: Analysis software
DNA oligo primers	IDT	Custom order	Protocol 2
LNA probes	IDT	Custom order	Protocol 2
Software tool	IDT	Web site: http://biophysics.idtdna.com/	Protocol 2
dbSNP database	NCBI	Web site: https://www.ncbi.nlm.nih.gov/projects/SNP/	Protocol 2
TE buffer	ThermoFisher SCIENTIFIC	12090015	Protocol 3
DNase/RNase-Free Distilled Water	ThermoFisher SCIENTIFIC	10977015	Protocol 3 (Table 1)
Clarity Digital PCR Probe Mastermix	JN Medsys	12013	Protocol 3 (Table 1): 2xPCR Master Mix
			A component of #10011
Clarity Sealing Fluid	JN Medsys	12005	Protocol 3: Sealing fluid
			A component of #10011
Clarity JN Solution	JN Medsys	12006	Protocol 3 (Table 1): 20xdPCR solution
			A component of #10011
Clarity Tube-strip	JN Medsys	12007	Protocol 3: Chip-in-a-tube
			A component of #10011
Clarity Sample Loading Kit	JN Medsys	12008	Protocol 3: Loading platform and slider
			A component of #10011
Clarity Auto Loader	JN Medsys	11002	Protocol 3: Auto loader
			A component of #10001
Clarity Sealing Enhancer	JN Medsys	11003	Protocol 3: Sealing enhancer
			A component of #10001
Clarity Reader	JN Medsys	11004	Protocol 3: Reader
			A component of #10001
Life Eco Thermal Cycler	Bioer Technology	TC-96GHbC	Protocol 3: Thermal cycler
Clarity Software	JN Medsys	Bundled with #10001	Protocol 3: Analysis software
High Sensitivity D1000 screen tape	Agilent Technologies	5067-5584	Protocol 4: Gel device for high sensitivity