18 July 2003 Instrumentation for continuous array genotyping of short insertion/deletion polymorphisms
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Abstract
Methods for DNA testing are abundant, however, there is still a need for lower cost, higher-throughput genotyping. Much emphasis has been placed on short tandem repeat polymorphisms (STRPs) and single nucleotide polymorphisms (SNPs). Another important polymorphism is the diallelic short insertion/deletion (indel). Our laboratory is using a continuous reel of 384 well arrays on polypropylene tape to genotype indel polymorphisms. The reel of arrays allows low cost automation, and an opportunity to decrease reaction volumes. The diallelic indel is typed by tagging allele-specific PCR primers with a FAM or JOE molecular beacon uniprimer. Our most recent array pattern contains wells that hold a maximum of 1.1 microliters with reaction volumes of 800 nanoliters. Because micro array tape (microtape) is unique, commercial equipment is not yet available. A series of instruments were developed in-house to handle the tape. A pipetting instrument was developed to deliver the DNA samples or other reagents. A solenoid micro-valve aspirating and jetting unit was developed for dispensing a common reaction mix. The arrays are sealed with commercially available microtiter plate seal material prior to polymerase chain reaction (PCR) within a custom built waterbath thermal cycler. The arrays are scanned using an epi-fluorescence detection unit designed to read FAM and JOE fluorescent dyes. The detector uses an argon ion laser for excitation and two photomultiplier tubes (PMTs) for detection. The resulting images are processed using custom software.
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Terry L. Rusch, Terry L. Rusch, William Dickinson, William Dickinson, Jian Che, Jian Che, Kim Fieweger, Kim Fieweger, Jon Chudyk, Jon Chudyk, Mitchel J. Doktycz, Mitchel J. Doktycz, Adong Yu, Adong Yu, James L. Weber, James L. Weber, } "Instrumentation for continuous array genotyping of short insertion/deletion polymorphisms", Proc. SPIE 4966, Microarrays and Combinatorial Technologies for Biomedical Applications: Design, Fabrication, and Analysis, (18 July 2003); doi: 10.1117/12.483474; https://doi.org/10.1117/12.483474
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