Digital Droplet Polymerase Chain Reaction for Indel Mutation Detection in Target Genes

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

1. 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.
2. Calculate the copy number using the haploid genome weight and concentration of DNA in the extract. This is done by multiplying the concentration of the extracted DNA by the volu....

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
<em><strong>Reagents</strong></em>
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
<em><strong>Materials</strong></em>
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.
<em><strong>Equipment</strong></em>
C1000 Touch Thermal Cycler	Bio-Rad	1851148	Can use other Thermo cycler with gradient function and deep well