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Biomolecular Research Facility

The DNA Sequencing and Genotyping Program under the Biomolecular Research Facility offers automated DNA sequencing and Genescan services. Specific genes can be sequenced as insert in a bacterial phage vector (single-stranded), plasmid vector, lambda vector, BAC or YAC (all double-stranded), or as a PCR product (direct sequencing). Currently, we utilize the BigDye Terminator from PE Biosystems. Typically with a clean template, double stranded and plasmid DNA will yield 750-800 bp with an accuarcy of 98%. Single stranded DNA can be sequenced up to the same length or longer with the same accuracy. For direct sequencing of PCR products the long templates can reach the similar specifications as the plasmid DNA while the short ones can have various degrees of accuracy beause of the affinities they possess for the DNA polymerase. For BAC or PAC DNAs, the specifications are unavailable at the time being.

Sequencing is routinely performed on an Applied Biosystems 377 Prism DNA Sequencer, using BigDye terminator chemistry with Taq DNA polymerase. This method involves the use of four fluorescent dye-labeled dideoxy nucleotides. All four termination reactions are performed in a single tube and the reaction products, after proper purification, are loaded in a single lane at a polyacrylamide slab gel for electrophoresis. Real time detection of the electrophoresed individual fragments is achieved with laser scanning in conjunction with CCD camera imaging production.

Other chemistries, such as dye primer sequencing, and selected chemistry with alternate enzymes, such as Sequenase, are also available upon request. Special arrangements can be made to switch chemistries upon request. DNA samples can be sequenced with primers provided by the users or requested from our Primer Bank. The bank was built with the intention of including as many commonly used primers as practical and will expand on reasonable suggestions.

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