Rapid multiplex single nucleotide polymorphism genotyping based on single base extension reactions and color-coded beads
ABSTRACT A single nucleotide polymorphism (SNP) typing method using color-coded beads is promising because it is easy to use and inexpensive. However, the present protocols are not suitable for clinical and diagnostic applications because they need centrifugation for bead-washing. Here, we developed a simplified protocol without a bead-washing procedure that enables SNP typing of PCR amplified fragments in only 30 min.
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ABSTRACT: This study reports the implementation of three strategies for the development of genetic markers and their evaluation in both progenitors of an F2 population used for the construction of a genetic map of Coffea arabica. The strategies were Cleaved Amplified Polymorphic Sequences (CAPS), Single Strand Conformational Polymorphism (SSCP), and sequence analysis predicted Single Nucleotide Polymorphism (SNP). The methodologies were developed from different sequence sources: For CAPS, we used 25 COS sequences derived from Hedyotis spp. and 29 COSII sequences derived from Solanaceae and Rubiaceae species; for SSCP, we used 111 coffee EST sequences, 50 COSII sequences, and 10 C. arabica BAC end sequences. A low polymorphism was identified with the CAPS and SSCP methodologies. A total of 61 SNPs were identified insilico from 5,371 ESTs of coffee and from amplified, cloned, and sequenced COSII markers. Sixteen of these SNPs were validated with Luminex technology and 2 of them were polymorphic in C. arabica genotypes. This study highlights the difficulties of finding polymorphism in the species C. arabica where SNP identification seems to be the best strategy to search for polymorphic markers for this low diversity plant. Keywords Coffea arabica -Molecular markers-Polymorphism-SNPs-CAPS-SSCPEuphytica 05/2009; 173(2):243-253. DOI:10.1007/s10681-009-0102-6