High-Performance PCR for Alleles Discrimination of Chromo-Helicase-DNA Binding Protein (CHD1) Gene in Bird Sexing
1
Institute for Biomedical Research and Innovation-National Research Council (IRIB-CNR), Via Ugo La Malfa 153, 90146 Palermo, Italy
2
Department of Biological, Chemical and Pharmaceutical Sciences and Technologies (STEBICEF), University of Palermo, Viale delle Scienze, bd. 16, 90128 Palermo, Italy
3
Department of Earth and Marine Sciences (DiSTeM), University of Palermo, Viale delle Scienze, bd. 16, 90128 Palermo, Italy
4
Ornis Italica, Piazza Crati 15, 00199 Roma, Italy
5
Department of Agriculture, Food and Forest Sciences (SAAF), University of Palermo, Viale delle Scienze, bd. 13, 90128 Palermo, Italy
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Biology 2023, 12(2), 300; https://doi.org/10.3390/biology12020300
Submission received: 2 December 2022
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Revised: 8 February 2023
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Accepted: 10 February 2023
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Published: 14 February 2023
(This article belongs to the Section Genetics and Genomics)
Simple Summary
Rapid results in genetic tests are often required, including in outdoor analyses. To date, the only fast technique is LAMP (loop-mediated isothermal amplification), which is overall less flexible than PCR. We have developed a PCR test in which very high DNA yields are achieved, allowing for direct in-tube detection of the products by fluorescence. We successfully tested this technique in rapid sexing of Procellariiformes as proof of concept, which paves the way for further development of the method to many applications.
Abstract
Genetic analyses aiming at assessing the presence of specific sequences or alleles are often carried out by PCR. Sexing of most birds is nowadays based on PCR with “universal” primers and relies on the assessment of the presence of the sex-linked CHD1-Z and -W alleles. The entire workflow is relatively time-consuming, especially for batch analyses, whereas methods that allow carrying out the entire procedure in a short time are highly desirable. The only method for outdoor analyses reported so far relies on LAMP; however; it fails to work properly in Procellariiformes. Besides improving the LAMP test; we have developed a PCR-based DNA amplification procedure (named high-performance PCR); whose unique features allow it to outperform standard PCR; making possible the direct, in-tube visual reading of results. We tested it with specifically designed Procellariiformes-targeted primer sets for rapid sexing of the birds using fluorimetric detection. The protocol, combined with rapid DNA extraction, allows for fast reading of results without electrophoresis within less than 1 h from sampling. The technique could be extended to other species, as well as to many other applications.
