Digital Droplet Polymerase Chain Reaction for Indel Mutation Detection in Target Genes
Published: May 31, 2023
Abstract
Source: Carballar-Lejarazú, R., et al. Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations. J. Vis. Exp. (2021).
This video demonstrates the role of digital droplet polymerase chain reaction in identifying indel mutation in a DNA sample. The high precision and sensitivity of ddPCR enable accurate and consistent detection of rare mutations present in a sample, even at low levels. It can identify and quantify indel mutations in different regions of the genome, providing valuable insights into the mechanisms of mutation and the functional consequences of these mutations.
Protocol
1. ddPCR reactions and droplet generation preparation Quantify DNA using a fluorometer. NOTE: For the drop-off assay, it is recommended to use a range of 3,000-30,000 haploid genome copies per reaction, which is designed to detect NHEJ events with a HEX-labeling probe that binds to a WT sequence of the targeted cut site and will not anneal (drop-off) if there is a deletion or insertion at the target site, indicating the presence of an NHEJ variant. Calculate the copy number u…
Representative Results
Figure 1: Experimental setup and procedure. (A) Cartridge preparation for Droplet generation. (Top) Samples are filled in the middle row of the cartridge, while oil is filled in the bottom row. (Bottom) Top row filled with emulsified droplets after droplet generation. (B) Droplet generator with a cartridge filled with sample and covered by a gasket in place. (<st…
Declarações
The authors have nothing to disclose.
Materials
| Reagents | |||
| ddPCR Super Mix for Probes (no dUTP) | Bio-Rad | 1863024 | |
| EDTA (pH 8.0) | Invitrogen | AM9260G | |
| PCR-grade Water | Any certified PCR-grade water can be used | ||
| Proteinase K 20 mg/mL | Thermo Fisher Scientific | AM2546 | |
| Materials | |||
| ddPCR 96-Well Plate | Bio-Rad | 12001925 | |
| Droplet Generator DG8 Cartridge and Gaskets | Bio-Rad | 1864007 | |
| Droplet Generation Oil for probes | Bio-Rad | 1863005 | |
| Fluorescent probes (e.g. FAM/HEX probes) | Sigma-Aldrich | N/A | Probes are experiment specific and can be purchased from any certified seller available. |
| Forward and Reverse oligonucleotide primers | Sigma-Aldrich | N/A | Primers are experiment specific and can be purchased from any certified seller available. |
| Equipment | |||
| C1000 Touch Thermal Cycler | Bio-Rad | 1851148 | Can use other Thermo cycler with gradient function and deep well |
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Citar este artigo
Digital Droplet Polymerase Chain Reaction for Indel Mutation Detection in Target Genes. J. Vis. Exp. (Pending Publication), e21352, doi: (2023).