Welcome to the
University of Virginia
DNA Sciences Core

 

 

 

Automated DNA Sequencing is an essential service by the DNA Sciences Core. We use the state-of -art capillary instrument, 3730 DNA Analyzer from the Applied Biosystems. Specific nucleic acid sequences can be determined 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). Genomic DNA or mitochondrial DNA can be sequenced directly of after certain derivatization.

 

Currently, we use the BigDye Terminator v3.1 chemistry from Applied Biosystems, which provides top performance under Sanger sequencing methods. Typically with a clean template with roughly balanced bases, double stranded and plasmid DNA will yield 750-800 bp with an accuracy of 98% or higher. 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 because of the varying affinities they possess for the DNA polymerase due to their lengths.

 

Ordering a sequencing run includes two steps. First, users are to fill out an Online Request Form. Unnecessary delay could result in if the form is not completed properly. The turnaround time is normally 24-36 hours for samples submitted before 10 AM. Users may pledge their case with us if they need expeditions for special occasions, such as a grant deadline.

 

For difficult-to-sequence templates we have established a regiment of methods to deal with but we need some prior knowledge of them to prescribe an effective recipe to use. The different approaches include the use of DMSO or betaine, or modification of the cycling profiles. In addition, modified nucleotides are sometimes used to alleviate the compression problems. We encourage users to consult with us when they encounter or anticipate difficult templates.

 

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.

 

You can now order oligos from the UVA-Invitrogen program (See the logo in the left panel) that may save you a lot in primer costs. It is convenient, quick, and less hassled -- You order your oligos online, NO PTAO is required. You can pick up the oligos in the core and the get billed only once a month, again online. You need to register to use this feature, please contact us if you need help.

 

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