Cost-effective method to synthesize a fluorescent internal DNA standard for automated fragment sizing.

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

Biotechniques

journal_title

BioTechniques

authors

Brondani RP,Grattapaglia D

doi

10.2144/01314st06

subject

Has Abstract

pub_date

2001-10-01 00:00:00

pages

793-5, 798, 800

issue

4

eissn

0736-6205

issn

1940-9818

journal_volume

31

pub_type

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