<abstract xmlns="http://www.w3.org/1999/xhtml"><p>Integration of fluorescent-dUTP in polymerase chain reaction (PCR) appears to be a sound method for fluorescence labelling of amplicons in genotyping with simple sequence repeats (SSRs) using an automated sequence analyser. However, the method has not been explored in terms of performance optimisation and cost control. In this paper, we optimised the protocol for fluorescent-dUTP based SSR genotyping in a case study with <italic>Eucalyptus</italic>. A combination of low dNTP concentration (25 μM each) in PCR reaction and a touchdown PCR programme contributed to increase dramatically the fluorescent intensity of SSR amplicons, thereby facilitating accurate and multiplexed scoring of SSR alleles. The usefulness of the optimised protocol was demonstrated in its application to genetic mapping of SSR loci onto <italic>E. urophylla</italic> and <italic>E. tereticornis</italic> linkage maps constructed previously. The protocol optimised here would provide a reliable and economical assay for sequencer-based SSR genotyping in a wide range of biological applications.</p></abstract>

Integration of fluorescent-dUTP in polymerase chain reaction (PCR) appears to be a sound method for fluorescence labelling of amplicons in genotyping with simple sequence repeats (SSRs) using an automated sequence analyser. However, the method has not been explored in terms of performance optimisation and cost control. In this paper, we optimised the protocol for fluorescent-dUTP based SSR genotyping in a case study with Eucalyptus. A combination of low dNTP concentration (25 μM each) in PCR reaction and a touchdown PCR programme contributed to increase dramatically the fluorescent intensity of SSR amplicons, thereby facilitating accurate and multiplexed scoring of SSR alleles. The usefulness of the optimised protocol was demonstrated in its application to genetic mapping of SSR loci onto E. urophylla and E. tereticornis linkage maps constructed previously. The protocol optimised here would provide a reliable and economical assay for sequencer-based SSR genotyping in a wide range of biological applications.

An Optimised Protocol for Fluorescent-dUTP Based SSR Genotyping and its Application to Genetic Mapping in <italic>Eucalyptus</italic>

Silvae Genetica

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

An Optimised Protocol for Fluorescent-dUTP Based SSR Genotyping and its Application to Genetic Mapping in 

Integration of fluorescent-dUTP in polymerase chain reaction (PCR) appears to be a sound method for fluorescence labelling of amplicons in genotyping with...

An Optimised Protocol for Fluorescent-dUTP Based SSR Genotyping and its Application to Genetic Mapping in Eucalyptus

Publicado en línea: 05 ago 2017

Páginas: 18 - 25

Recibido: 12 ene 2010

DOI: https://doi.org/10.1515/sg-2011-0003

Palabras clave
Simple sequence repeats (SSRs), genotyping, fluorescent-dUTP, genetic mapping

© 2011 F. Li et al., published by De Gruyter Open

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

An Optimised Protocol for Fluorescent-dUTP Based SSR Genotyping and its Application to Genetic Mapping in Eucalyptus

Publicado en línea: 05 ago 2017

Páginas: 18 - 25

Recibido: 12 ene 2010

DOI: https://doi.org/10.1515/sg-2011-0003

Palabras claveSimple sequence repeats (SSRs), genotyping, fluorescent-dUTP, genetic mapping

© 2011 F. Li et al., published by De Gruyter Open

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License.

Palabras clave
Simple sequence repeats (SSRs), genotyping, fluorescent-dUTP, genetic mapping