PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.
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.
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 zebrafish3,4. These techniques can efficiently generate genetically modified animals, with high-frequency mutation creation and germ-line transmission3,4.
Despite these advances, traditional genotyping techniques in zebrafish limit the full power of the mutagenesis and transgenesis tools. PCR followed by gel electrophoresis, sometimes combined with restriction enzyme digestion, is widely used to detect genome modification, but is time-consuming and less sensitive to identify small insertions or deletions. TaqMan probe assays have high initial costs and require careful optimization. Sequencing of PCR products can take several days and is not practical for large-scale screening. Restriction fragment length polymorphism (RFLP) analysis can only discriminate SNPs affecting a limited range of restriction enzyme recognition sites.
High-resolution melting analysis (HRMA), a closed-tube post-PCR analysis method, is a recently developed method that is rapid, sensitive, inexpensive, and amenable to screening large numbers of samples. HRMA can be used to detect SNPs, mutations, and transgenes5-7. HRMA is based on thermal denaturation of double-stranded DNAs, and each PCR amplicon has a unique dissociation (melt) characteristic5. Samples can be discriminated due to their different nucleotide composition, GC content, or length, typically in combination with a fluorescent dye that only binds double-stranded DNA8. Thus, HRMA can distinguish different genotypes based on the different melt-curve characteristics. Because HRMA uses low-cost reagents and is a single-step post-PCR process, it can be used for high-throughput strategies. HRMA is nondestructive, so following analysis the PCR amplicons can be used for other applications. HRMA has been applied in many organisms and systems, including cell lines, mice, and humans9-11. Its use has recently been described in zebrafish to detect mutations induced by zinc finger nucleases (ZFNs) and TALENs6,12,13.
In this paper, we describe how to perform PCR-based HRMA in embryonic and adult zebrafish (Figure 1). This protocol is suitable for detecting SNPs, transgenes, and mutations, including single base-pair changes, insertions, or deletions.
1. DNA Preparation
2. PCR
3. HRMA
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 normalization of the variation of the different sample curves, using pre- and post-melt regions as standards (Figure 2C). This improves comparison of results from different samples in which the variation of fluorescence is related to minor experimental variations. Each pair of temperature lines for normalization should be placed approximately 1 ˚C apart (Figure 2C, arrowheads). Grouping of data results is represented in two different ways: an upper graph that shows melt curve profiles, and a lower graph that shows a subtractive difference plot in comparison to a reference sample (Figure 2D).
HRMA analysis can be used to detect mutations (Figures 3A and 3B) or transgenes (Figure 3C). In Figure 3A, two different mutations in eif2b5 gene are shown (red and blue curves in Figure 3A) by subtracting the normalized fluorescence data of the wild-type sample. It does not matter which genotype is chosen for reference. More subtle or confusing differences in melt-curves can be distinguished by using the subtractive difference plot. In Figure 3B, an example of four different genotypes in a single collection of embryos is shown.
Figure 1. Outline of protocol for PCR-based HRMA of zebrafish genomic DNA. DNA extraction from whole tissue (fin-clip or embryo) is followed by PCR amplification in the presence of a fluorescent double-stranded DNA-binding dye. The PCR amplicon is denatured and fluorescence signal is recorded, followed by melt-curve analysis, to detect the amplicon and/or to differentiate genotypes.
Figure 2. Screen-shots of software to perform HRMA and analyze results. A. Screen shot of LightScanner software; yellow box is magnified in “B”. B. Magnified view of boxed region in “A”. Setting Start and End Temp are shown (arrows). C. Position the premelt and post-melt parallel lines for normalization (arrowheads). Notice the fluorescence variance in pre- and post-melting regions (red box). Lower graph (green box) shows melt-curves derived from the raw data plots following normalization. D. Upper graph shows melt curve profiles after automatic grouping; different genotypes are illustrated in different colors (red box). Lower graph (green box) shows the fluorescence difference curve. Click here to view larger image.
Figure 3. Using HRMA to identify mutations and transgenes. Fluorescence difference curves are shown; change in fluorescence (y-axis) to temperature (x-axis) is shown. A. HRMA was used to detect fish carrying mutations in the eif2b5 gene. Wild type is shown in grey (arrow). Red and blue colors represent two different eif2b5 mutations, confirmed by sequencing of the PCR product. B. Four different foxP2 mutant alleles are identified with HRMA. Red: zc82/+ (8 bp deletion), green: zc83/+ (17 bp deletion), blue: zc82/zc83 (8bp deletion/17bp deletion), grey: zc83/zc83 (17bp deletion homozygotes). C. Gal4 transgenic fish (grey curves) can be distinguished from wild type (brown curve, arrow). Notice that for transgene detection, normalization should not be performed. Click here to view larger image.
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.
A critical step for this technique is PCR amplicon and primer design. Typical length for PCR products in HRMA is 50-200 bp. Short amplicons increase sensitivity and are ideal for detecting single nucleotide changes. If melt-curves of closely related PCR product species can not be distinguished easily, a small oligonucleotide complementary to the SNP and blocked at the 3’ end can be included in the PCR reaction (Lunaprobes; probes). The probes are generated by asymmetric PCR using different concentration of the forward and reverse primers, with a 3’ C3 blocker to prevent probe extension in the subsequent HRMA PCR. The probe is then included in the HRMA PCR reaction. HRMA then occurs with the probe, in which the asymmetric probe binds to the PCR product and generates probe-target duplexes with different melt-curves that can be used to distinguish alleles.
Several companies offer melt-curve analysis systems. These include the Biofire Lightscanner, the Roche LightcyclerVR 480 and the Qiagen Rotor-Gene Q. Several fluorescent, DNA-binding dyes are available, including LC Green PLUS and SYT09 EvaGreen. There is some variation in sensitivity in mutation detection based on different fluorescent dyes and melt-curve machines8,15. Nonsaturating dyes, for example, SYBR Green I, are unsuitable for most HRMA applications: at high concentrations, SYBR Green I inhibits the activity of DNA polymerase; and at lower concentrations, SYBR Green I cannot precisely measure melting behavior due to its redistribution from melted regions back to regions of dsDNA16. In general most existing Real Time PCR platforms are capable of preforming HRMA using a software package add-on and a second-generation fluorescent DNA binding dye. While technical details of the software and sequence of experimental steps vary slightly between platforms, the overall scientific concepts are essentially identical.
Our lab and others have demonstrated that HRMA can be used in both embryonic and adult zebrafish to detect transgenes, and mutations induced by ZFNs and TALENs6,12,13. HRMA has been used to discover polymorphisms in zebrafish6,12. Our lab uses HRMA both for initial screening of TALEN-induced mutations as well as for sorting of established lines (TSQ and JLB, unpublished). Other potential applications that could be applied to zebrafish include methylation-sensitive HRMA (ms-HRMA)17, quantification of copy-number variants5; and pathogen detection in vivariums18.
The authors have nothing to disclose.
We thank members of the Blaschke, Grunwald, and Wittwer labs for advice and technical assistance. This work is supported by the PCMC Foundation, NIH R01 MH092256 and DP2 MH100008, and the March of Dimes Foundation research grant #1-FY13-425, to JLB.
100 Reaction LightScanner Master Mix | BioFire | HRLS-ASY-0002 | www.biofiredx.com | Store at -20 °C |
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 |