Development of UCDS
Bennett's goal was to develop a mutation-detection procedure that he named universal condition direct sequencing (UCDS), in which universal conditions for both the PCR and the sequencing reactions would optimize the sequencing data, simplify the molecular identification, and lower the cost of the research procedure.
A collaboration was formed between Dr. Kunkels lab and Applied Biosystems. Soon after, Children's Hospital became a testing facility for research in the use of VariantSEQr primers for gene sequence variations associated with muscular dystrophy.
Today, the sequence for the dystrophin gene, all of the reported regions of interest, including all the exons, is 100% covered by a combination of VariantSEQr primers and about 20 primers designed by Bennett. Together, Dr. Kunkel's research lab and Applied Biosystems have now also designed assays to discover variants in 10 other muscular dystrophy-associated genes and are working on the remaining 30. These assays should be of great help to researchers in their efforts to find causes and cures for muscular dystrophy.
Instead of designing PCR primers, calculating melting temperatures (TMs), and then assigning primers to groups based on similar TMs for the UCDS technique, Bennett has designed sets of primers for each assay that amplify target sequences under identical PCR conditions.
“What makes the UCDS process universal is that we get everything to work at the same temperature. We place all of the DNA samples in a single thermal cycler reaction plate and amplify all of them at the same time on the same instrument instead of in groups. We then sequence all of the samples using universal sequencing primers,“ explains Bennett.
Using the UCDS process, Bennett can sequence any amplified DNA region by using the M13 forward or M13 reverse universal primers.
“A universal condition for all of the sequencing reactions improves your chances of obtaining good sequencing data. With universal primers instead of unique primers for each cycle-sequencing reaction you do not need to have a different set of rules for each set of primers.
“After you do your PCR, you add the BigDye Terminator Cycle Sequencing reagents and the M 13 forward or M 13 reverse universal primers to a purified and normalized amount of PCR product, run your cycle-sequencing reactions, and prepare them for sequencing on the Applied Biosystems' 3730 DNA Analzyer,“ says Bennett.
As part of the integrated workflow of the VariantSEQr Resequencing System, sequence files containing base calls of sample DNA sequences are exported from the 3730 system to the SeqScape software application. SeqScape software simplifies identification of gene sequence variants by comparing sample DNA sequences to a consensus sequence of the gene being resequenced.
The SeqScape software that Bennett uses includes the consensus sequence for the gene under study. For the entire sequence of regions of interest, including all exons of the gene, SeqScape software identifies the most common base found at each position along the stretch of DNA that makes up the gene.
Using SeqScape software, researchers tab through the consensus sequence, as well as through their sample sequence, one on top of the other in the display. The tab stops at every base position where there is a known variation in the consensus sequence or at a position where the sample DNA sequence has a single base that differs from the consensus sequence.
“When I analyze muscular dystrophy DNA samples using the SeqScape software, I tab through the DNA sequence and in three minutes, I find a mutation that could have taken me weeks or months to locate before I had the software,“ notes Bennett.
In the design of new assays for UCDS, Bennett uses the SeqScape consensus templates to identify exons and decide which regulatory sequences—introns, splice sites, promoters—and UTRs (untranslated regions) to include in his primer design plans.
According to Bennett, the major cost savings associated with using the UCDS process for the discovery of gene sequence variants will come from reducing the volume of reagents used for the PCR. Bennett hopes to reduce PCR reagent volume from 25 microliters per reaction to six microliters.
Additional cost savings are possible by automating the process further through the use of a robotic device to dispense liquids for the reactions, to normalize and purify the PCR reactions, as well as to set up and purify the cycle-sequencing reactions and prepare them for the 3730 DNA Analyzer. This speeds the entire process and reduces time and labor costs.