Abstract:
:We describe a simple and cost-effective method for the synthesis of an internal fluorescently labeled DNA standard for fragment sizing using an automated DNA sequencer. A set of primer pairs labeled with ROX was developed to amplify 12 DNA fragments, 58-417 bp, derived from a conserved region of plant chloroplast DNA. These amplified fragments were mixed together, constituting a fluorescent internal DNA size marker. The precision of the size standard was evaluated by estimating the size of 20 alleles that were amplified at four dinucleotide microsatellite loci with the synthesized size standard and the commercial internal sizing standard, GeneScan Rox500. A number of intra-gel and inter-gel comparisons were run, and an analysis of variance was carried out. No significant difference was observed between the size estimates obtained with the synthesized DNA standard and the commercial standard. This facile and general PCR-based method for the synthesis of internal standards allows for significant savings in the implementation of large genotyping experiments using microsatellite or AFLP markers.
journal_name
Biotechniquesjournal_title
BioTechniquesauthors
Brondani RP,Grattapaglia Ddoi
10.2144/01314st06subject
Has Abstractpub_date
2001-10-01 00:00:00pages
793-5, 798, 800issue
4eissn
0736-6205issn
1940-9818journal_volume
31pub_type
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