Journal ArticleDOI
Universal primers for fluorescent labelling of PCR fragments--an efficient and cost-effective approach to genotyping by fluorescence.
TLDR
A suite of four high‐performance universal primers that can be employed in a three primer PCR approach for efficient and cost‐effective fluorescent end‐labelling of PCR fragments and are combined with multiple fluorophores to co‐amplify multiple loci efficiently via multiplex PCR.Abstract:
Directly labelling locus-specific primers for microsatellite analysis is expensive and a common limitation to small-budget molecular ecology projects. More cost-effective end-labelling of PCR products can be achieved through a three primer PCR approach, involving a fluorescently labelled universal primer in combination with modified locus-specific primers with 5' universal primer sequence tails. This technique has been widely used but has been limited largely due to a lack of available universal primers suitable for co-amplifying large numbers of size overlapping loci and without requiring locus-specific PCR conditions to be modified. In this study, we report a suite of four high-performance universal primers that can be employed in a three primer PCR approach for efficient and cost-effective fluorescent end-labelling of PCR fragments. Amplification efficiency is maximized owing to high universal primer Tm values (approximately 60+ °C) that enhance primer versatility and enable higher annealing temperatures to be employed compared with commonly used universal primers such as M13. We demonstrate that these universal primers can be combined with multiple fluorophores to co-amplify multiple loci efficiently via multiplex PCR. This method provides a level of multiplexing and PCR efficiency similar to microsatellite fluorescent detection assays using directly labelled primers while dramatically reducing project costs. Primer performance is tested using several alternative PCR strategies that involve both single and multiple fluorophores in single and multiplex PCR across a wide range of taxa.read more
Citations
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Journal ArticleDOI
Environmental monitoring using next generation sequencing: rapid identification of macroinvertebrate bioindicator species
TL;DR: Next generation sequencing using two genes was successful for identifying chironomid species, however, when detecting species from 454 pyrosequencing data sets it was critical to include known individuals for quality control and to establish thresholds for detecting species.
Journal ArticleDOI
Strong genetic structure corresponds to small-scale geographic breaks in the Australian alpine grasshopper Kosciuscola tristis
Rachel A. Slatyer,Michael A. Nash,Michael A. Nash,Adam D. Miller,Yoshinori Endo,Yoshinori Endo,Kate D. L. Umbers,Kate D. L. Umbers,Ary A. Hoffmann +8 more
TL;DR: Testing the hypothesis that genetic partitioning reflects observable geographic structuring across Australia’s mainland high country, in the widespread alpine grasshopper Kosciuscola tristis found broadly congruent patterns of regional structure between the DNA sequence and microsatellite datasets corresponding to strong divergence among isolated mountain regions.
Journal ArticleDOI
Genome-wide SNPs lead to strong signals of geographic structure and relatedness patterns in the major arbovirus vector, Aedes aegypti
TL;DR: The new SNP set provided much greater resolution in detecting population structure and estimating fine-scale relatedness than a set of polymorphic microsatellites, critical for implementing new control measures against this major disease vector Ae.
Journal ArticleDOI
The genome and linkage map of the northern pike (Esox lucius): conserved synteny revealed between the salmonid sister group and the Neoteleostei.
Eric Rondeau,David R. Minkley,Jong S. Leong,Amber M. Messmer,Johanna R. Jantzen,Kristian R. von Schalburg,Craig Lemon,Nathan H. Bird,Ben F. Koop +8 more
TL;DR: The first genome assembly, reference transcriptome and linkage map for northern pike is presented and the suitability of this species to provide a representative pre-duplication genome for future studies of salmonid and teleost evolution is evaluated.
Journal ArticleDOI
Genetic rescue increases fitness and aids rapid recovery of an endangered marsupial population.
Andrew Weeks,Dean Heinze,Louise Perrin,Jakub Stoklosa,Ary A. Hoffmann,Anthony van Rooyen,Thomas Kelly,Ian Mansergh +7 more
TL;DR: Increased hybrid fitness in a rescued population of the mountain pygmy possum is reported, which likely contributed to population growth following genetic rescue, and points to genetic rescue as being a potentially useful option for the recovery of small threatened populations.
References
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Genome sequencing in microfabricated high-density picolitre reactors
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An economic method for the fluorescent labeling of PCR fragments
TL;DR: A poor man's approach to genotyping for research and high-throughput diagnostics and how it can be applied to medicine and science.
Journal ArticleDOI
OligoCalc: an online oligonucleotide properties calculator.
TL;DR: OligoCalc incorporates three common methods for calculating oligonucleotide-melting temperatures, including a nearest-neighbor thermodynamic model for melting temperature.
Journal ArticleDOI
Current trends in microsatellite genotyping.
Erwan Guichoux,Lélia Lagache,Stefanie Wagner,Stefanie Wagner,Philippe Chaumeil,Patrick Léger,Olivier Lepais,Olivier Lepais,Camille Lepoittevin,Thibaut Malausa,E. Revardel,Franck Salin,Rémy J. Petit +12 more
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