Universal primers for fluorescent labelling of PCR fragments- an efficient and cost-effective approach to genotyping by fluorescence

Blacket, M.J., Robin, C., Good, R.T., Lee, S.F. and Miller, A.D. 2012, Universal primers for fluorescent labelling of PCR fragments- an efficient and cost-effective approach to genotyping by fluorescence, Molecular ecology resources, vol. 12, no. 3, pp. 456-463, doi: 10.1111/j.1755-0998.2011.03104.x.

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Title Universal primers for fluorescent labelling of PCR fragments- an efficient and cost-effective approach to genotyping by fluorescence
Author(s) Blacket, M.J.
Robin, C.
Good, R.T.
Lee, S.F.
Miller, A.D.ORCID iD for Miller, A.D. orcid.org/0000-0002-1632-7206
Journal name Molecular ecology resources
Volume number 12
Issue number 3
Start page 456
End page 463
Total pages 8
Publisher Blackwell Publishing
Place of publication Chichester, Eng.
Publication date 2012-05
ISSN 1755-098X
1755-0998
Keyword(s) fluorescent labelling
genotyping
microsatellites
multiplex PCR
tailed primers
universal primers
Summary 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.
Language eng
DOI 10.1111/j.1755-0998.2011.03104.x
Field of Research 060411 Population, Ecological and Evolutionary Genetics
050199 Ecological Applications not elsewhere classified
Socio Economic Objective 970106 Expanding Knowledge in the Biological Sciences
HERDC Research category C1.1 Refereed article in a scholarly journal
ERA Research output type C Journal article
Copyright notice ©2012, Blackwell Publishing
Persistent URL http://hdl.handle.net/10536/DRO/DU:30088773

Document type: Journal Article
Collections: School of Ecology and Environment
Centre for Integrative Ecology
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