Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Summary

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

Abstract

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA). This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts. Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

Introduction

Zebrafish (Danio rerio) is a vertebrate model system widely used for studies of development and disease modeling. Recently, numerous transgenic and mutation technologies have been developed for zebrafish. Rapid transgenesis techniques, usually based on a Tol2 transposon system¹, have been combined with improved cloning options for multiple DNA fragment assembly². Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been used to target loci in both somatic and germline cells in zebrafish^3,4. These techniques can efficiently generate genetically modified animals, with high-frequency....

Protocol

1. DNA Preparation

1. Prepare DNA lysis buffer: 50 mM KCl, 10 mM Tris-HCl pH 8.3, 0.3% Tween 20, 0.3% NP40. Add fresh Proteinase K to a final concentration of 1 mg/ml on the day of use¹².
2. Tissue collection:
   1. For adult fish fin clip:
      1. Anesthetize fish: Place the fish in 0.004% MS-222 (tricaine) solution. Wait until gill movement slows.
      2. Put fish on a stack of 5-10 Kimwipes and cut a small piece of the tail fin, about 2-3 mm, with a sterile razor blade.
      3. Quickly place the fish in a labeled tank with fresh water for recovery; carefully label both the tank and corresponding tube that will contain ....

Results

The protocol can be performed during a single day or separated in steps over several days (flow diagram of work is shown in Figure 1). DNA extraction is followed by the melt and analysis of PCR amplicons. The temperatures for the melt of the amplicon depend on the size and GC-content, but generally start and end temperatures of 50 ˚C and 95 ˚C are appropriate (Figures 2A and 2B). Once the melt is performed, analysis of the fluorescence melt curves typically requires norm.......

Discussion

PCR combined with HRMA is a powerful technology for zebrafish genotyping. The advantages of this approach are its speed, robustness, and sensitivity to detect even point mutations. The entire protocol, from fin-clip to melt-curve analysis, can be performed in less than eight hours by a single individual. In addition, the technique is amenable for high-throughput screening; does not require the use of ethidium bromide; and is sealed for all PCR and analysis steps which helps minimize contamination issues.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100 Reaction LightScanner Master Mix	BioFire	HRLS-ASY-0002	www.biofiredx.com
Hard-Shell PCR 96-well BLK/WHT Plates	Bio-Rad Laboratories	HSP9665	www.bio-rad.com
Microseal 'B' Adhesive Seals	Bio-Rad Laboratories	MSB1001	www.bio-rad.com
96-well LightScanner Instrument	BioFire	LSCN-ASY-0040	www.biofiredx.com
LightScanner Software with Call-IT 2.0	BioFire		www.biofiredx.com
High-resolution Melting Analysis 2.0	BioFire		www.biofiredx.com
LightScanner Primer Design Software	BioFire		www.biofiredx.com
Vector NTI Software	Invitrogen		www.invitrogen.com
Tricaine
Paraformaldehyde