Rapid Multiplex Single Nucleotide Polymorphism Genotyping Based on Single Base Extension Reactions and Color-Coded Beads
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-0012, Japan. Journal of Bioscience and Bioengineering
(Impact Factor: 1.88).
02/2002; 94(4):368-70. DOI: 10.1016/S1389-1723(02)80180-3
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.
Available from: Lisa Seeb
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ABSTRACT: The hybridization reaction kinetics of DNA probes on beads arrayed in a capillary was investigated experimentally and theoretically by using fluid mechanical methods. A device was prepared to contain DNA probes conjugated on 103-microm-diameter beads that were queued in a 150-microm-diameter capillary. The hybridization experiments were performed by introducing sample into the capillary and moving it with a one-way or a reciprocal flow. From the relation between Reynolds number and the resistance coefficient of the system, we found that the flow in the system was turbulent and not laminar as has been said of other microfluidic devices. The reaction efficiency was estimated using a mass-transfer coefficient derived from Chilton-Colburn's analogy. The estimate agreed well with the experimental data. A diffusion equation under laminar assumption was also solved, but this estimated value was 4.0-10.4 times smaller than the experimental data. Using the device achieved a hybridization efficiency as high as approximately 90% in 10 min. It was concluded that the high hybridization performance of the device resulted from turbulent flow and that the flow compensated the slow molecular diffusion. Using this bead-included structure resulted in a rapid and effective reaction at the solid-liquid interface, and the device seems very promising for many future applications.
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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.
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