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

Xing, Lingyan; Quist, Tyler S.; Stevenson, Tamara J.; Dahlem, Timothy J.; Bonkowsky, Joshua L.

doi:10.3791/51138

Cited by 29 publications

(26 citation statements)

References 18 publications

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“…PCR and high-resolution melt analysis (HRMA) was used for genotyping following published conditions [ 12 ] with LightScanner Master Mix (Biofire Defense, Inc.) with the following modification: 5 μL of the fluid collected from the Zebrafish Embryo Genotyper (ZEG) (from 11 μL total volume) was used in an 11 μL volume PCR. We used previously reported primers and conditions for PCR of the sa509 and zc90 alleles [ 11 ].…”

Section: Methodsmentioning

confidence: 99%

An automated system for rapid cellular extraction from live zebrafish embryos and larvae: Development and application to genotyping

et al. 2018

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Zebrafish are a valuable model organism in biomedical research. Their rapid development, ability to model human diseases, utility for testing genetic variants identified from next-generation sequencing, amenity to CRISPR mutagenesis, and potential for therapeutic compound screening, has led to their wide-spread adoption in diverse fields of study. However, their power for large-scale screens is limited by the absence of automated genotyping tools for live animals. This constrains potential drug screen options, limits analysis of embryonic and larval phenotypes, and requires raising additional animals to adulthood to ensure obtaining an animal of the desired genotype. Our objective was to develop an automated system that would rapidly obtain cells and DNA from zebrafish embryos and larvae for genotyping, and that would keep the animals alive. We describe the development, testing, and validation of a zebrafish embryonic genotyping device, termed “ZEG” (Zebrafish Embryo Genotyper). Using microfluidic harmonic oscillation of the animal on a roughened glass surface, the ZEG is able to obtain genetic material (cells and DNA) for use in genotyping, from 24 embryos or larvae simultaneously in less than 10 minutes. Loading and unloading of the ZEG is performed manually with a standard pipette tip or transfer pipette. The obtained genetic material is amplified by PCR and can be used for subsequent analysis including sequencing, gel electrophoresis, or high-resolution melt-analysis. Sensitivity of genotyping and survival of animals are both greater than 90%. There are no apparent effects on body morphology, development, or motor behavior tests. In summary, the ZEG device enables rapid genotyping of live zebrafish embryos and larvae, and animals are available for downstream applications, testing, or raising.

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Section: Methodsmentioning

confidence: 99%

An automated system for rapid cellular extraction from live zebrafish embryos and larvae: Development and application to genotyping

et al. 2018

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“… Since the identification of mutant alleles relies on the absence of a restriction site, including a wild-type control in the genotyping analysis may be a useful positive control. The protocol details a genotyping strategy based on RFLP, but alternate methods such as derived Cleaved Amplification Polymorphic Sequence (dCAPS; [ 10 ]) or High Resolution Melting Analysis (HRMA; [ 11 ]) genotyping assays could be used after the DNA extraction procedure. …”

Section: Dna Extraction From Tail Biopsies and Genotypingmentioning

confidence: 99%

“…The protocol details a genotyping strategy based on RFLP, but alternate methods such as derived Cleaved Amplification Polymorphic Sequence (dCAPS; [ 10 ]) or High Resolution Melting Analysis (HRMA; [ 11 ]) genotyping assays could be used after the DNA extraction procedure.…”

Section: Dna Extraction From Tail Biopsies and Genotypingmentioning

confidence: 99%

Combining genotypic and phenotypic analyses on single mutant zebrafish larvae

et al. 2018

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“…6 The majority of these fish are small bodied, genome-sequenced model species that are suitable for biomedical and ecological study, such as the zebrafish Danio rerio 7 and three-spined stickleback Gasterosteus aculeatus. 8 The frequent use of these species in genetic studies means that DNA samples often need to be obtained in vivo , usually by fin clipping under nonterminal anesthesia 9 ; however, the use of anesthetics may affect behavior and physiology. 10–12 Furthermore, within the scientific community there is an increased awareness that fish may experience pain, stress, or lasting harm as a consequence of invasive procedures, 3 , 11 driving the development of alternative methods.…”

Section: Introductionmentioning

confidence: 99%

A Low-Cost Method of Skin Swabbing for the Collection of DNA Samples from Small Laboratory Fish

et al. 2017

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Fin clipping of live fish under anesthesia is widely used to collect samples for DNA extraction. An alternative, potentially less invasive, approach involves obtaining samples by swabbing the skin of nonanesthetized fish. However, this method has yet to be widely adopted for use in laboratory studies in the biological and biomedical sciences. Here, we compare DNA samples from zebrafish Danio rerio and three-spined sticklebacks Gasterosteus aculeatus collected via fin clipping and skin swabbing techniques, and test a range of DNA extraction methods, including commercially available kits and a lower-cost, in-house method. We verify the method for polymerase chain reaction analysis, and examine the potential risk of cross contamination between individual fish that are netted together. We show that swabbing, which may not require the use of anesthesia or analgesics, offers a reliable alternative to fin clipping. Further work is now required to determine the relative effects of fin clipping and swabbing on the stress responses and subsequent health of fish, and hence the potential of swabbing as a refinement to existing DNA sampling procedures.

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

Cited by 29 publications

References 18 publications

An automated system for rapid cellular extraction from live zebrafish embryos and larvae: Development and application to genotyping

An automated system for rapid cellular extraction from live zebrafish embryos and larvae: Development and application to genotyping

Combining genotypic and phenotypic analyses on single mutant zebrafish larvae

A Low-Cost Method of Skin Swabbing for the Collection of DNA Samples from Small Laboratory Fish

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