Robust allele-specific polymerase chain reaction markers developed for single nucleotide polymorphisms in expressed barley sequences

Bundock, P. C., Cross, M. J., Shapter, F. M. and Henry, R. J. (2006) Robust allele-specific polymerase chain reaction markers developed for single nucleotide polymorphisms in expressed barley sequences. Theoretical and Applied Genetics, 112 2: 358-365. doi:10.1007/s00122-005-0137-6


Author Bundock, P. C.
Cross, M. J.
Shapter, F. M.
Henry, R. J.
Title Robust allele-specific polymerase chain reaction markers developed for single nucleotide polymorphisms in expressed barley sequences
Journal name Theoretical and Applied Genetics   Check publisher's open access policy
ISSN 0040-5752
1432-2242
Publication date 2006-01
Sub-type Article (original research)
DOI 10.1007/s00122-005-0137-6
Volume 112
Issue 2
Start page 358
End page 365
Total pages 8
Place of publication Heidelberg, Germany
Publisher Springer
Language eng
Formatted abstract
Many methods have been developed to assay for single nucleotide polymorphisms (SNPs), but generally these depend on access to specialised equipment. Allele-specific polymerase chain reaction (AS-PCR) is a method that does not require specialised equipment (other than a thermocycler), but there is a common perception that AS-PCR markers can be unreliable. We have utilised a three primer AS-PCR method comprising of two flanking-primers combined with an internal allele-specific primer. We show here that this method produces a high proportion of robust markers (from candidate allele specific primers). Forty-nine inter-varietal SNP sites in 31 barley (Hordeum vulgare L.) genes were targeted for the development of AS-PCR assays. The SNP sites were found by aligning barley expressed sequence tags from public databases. The targeted genes correspond to cDNAs that have been used as restriction fragment length polymorphic probes for linkage mapping in barley. Two approaches were adopted in developing the markers. In the first approach, designed to maximise the successful development of markers to a SNP site, markers were developed for 18 sites from 19 targeted (95% success rate). With the second approach, designed to maximise the number of markers developed per primer synthesised, markers were developed for 18 SNP sites from 30 that were targeted (a 60% success rate). The robustness of markers was assessed from the range of annealing temperatures over which the PCR assay was allele-specific. The results indicate that this form of AS-PCR is highly successful for the development of robust SNP markers. © Springer-Verlag 2005.
Keyword Hordeum-vulgare l
PCR
Amplification
Dna
Identification
Plants
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status Unknown

Document type: Journal Article
Sub-type: Article (original research)
Collections: Queensland Alliance for Agriculture and Food Innovation
ERA 2012 Admin Only
 
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Created: Wed, 09 Feb 2011, 12:01:52 EST