Hybridization and Selective Release of DNA Microarrays

Beer, N R ; Baker, B ; Piggott, T ; Maberry, S ; Hara, C M ; DeOtte, J ; Benett, W ; Mukerjee, E ; Dzenitis, J ; Wheeler, E K

Lawrence Livermore National Laboratory

关键词: Lawrence Livermore National Laboratory;    Dna;    Security;    Monitoring;    Pathogens;   

DOI  :  10.2172/1033734
RP-ID  :  LLNL-TR-519311
RP-ID  :  W-7405-ENG-48
RP-ID  :  1033734

美国|英语

来源: UNT Digital Library

【 摘 要 】

DNA microarrays contain sequence specific probes arrayed in distinct spots numbering from 10,000 to over 1,000,000, depending on the platform. This tremendous degree of multiplexing gives microarrays great potential for environmental background sampling, broad-spectrum clinical monitoring, and continuous biological threat detection. In practice, their use in these applications is not common due to limited information content, long processing times, and high cost. The work focused on characterizing the phenomena of microarray hybridization and selective release that will allow these limitations to be addressed. This will revolutionize the ways that microarrays can be used for LLNL's Global Security missions. The goals of this project were two-fold: automated faster hybridizations and selective release of hybridized features. The first study area involves hybridization kinetics and mass-transfer effects. the standard hybridization protocol uses an overnight incubation to achieve the best possible signal for any sample type, as well as for convenience in manual processing. There is potential to significantly shorten this time based on better understanding and control of the rate-limiting processes and knowledge of the progress of the hybridization. In the hybridization work, a custom microarray flow cell was used to manipulate the chemical and thermal environment of the array and autonomously image the changes over time during hybridization. The second study area is selective release. Microarrays easily generate hybridization patterns and signatures, but there is still an unmet need for methodologies enabling rapid and selective analysis of these patterns and signatures. Detailed analysis of individual spots by subsequent sequencing could potentially yield significant information for rapidly mutating and emerging (or deliberately engineered) pathogens. In the selective release work, optical energy deposition with coherent light quickly provides the thermal energy to single spots to release hybridized DNA. This work leverages LLNL expertise in optics, microfluids, and bioinformatics.