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- Title
Development and validation of a universal primer pair for the taxonomic and phylogenetic studies of vertebrates.
- Authors
Jafar, Sana; Anjum, Khalid Mahmood; Zahoor, Muhammad Yasir; Shehzad, Wasim; Naseem, Asif; Imran, Muhammad
- Abstract
Background: Recent studies in the field of molecular identification have described 16S rRNA gene as a highly informative fragment of mitochondrial DNA for species discrimination. This study presents a newly developed universal primer pair yielding an approximately 350 bp fragment of mitochondrial 16S rRNA, variable enough to encompass and identify all vertebrate classes. Methods and results: The primers were designed by aligning and analyzing over 1500 16S rRNA sequences downloaded from the NCBI nucleotide database. A total of 93 vertebrate species, spanning 27 orders and 55 families, were PCR-amplified to validate the primers. All the target species were successfully amplified and identified when aligned with reference sequences from the NCBI nucleotide database. Using the Kimura 2-parameter model, low intra-species genetic divergence of the target region was observed – from 0 to 4.63%, whereas relatively higher inter-species genetic divergence was observed, ranging from 4.88% to 69.81%. Moreover, the newly developed primers were successfully applied to a direct PCR protocol, making the workflow very cost-effective, time-saving and less laborious in comparison to conventional PCR. Conclusions: The short length, high variability and conserved priming sites of the target fragment across all vertebrate species make it a highly desirable marker for species identification and discrimination.
- Subjects
IDENTIFICATION; VERTEBRATES; DATABASES; RIBOSOMAL RNA
- Publication
Molecular Biology Reports, 2024, Vol 51, Issue 1, p1
- ISSN
0301-4851
- Publication type
Article
- DOI
10.1007/s11033-023-09175-w