One reason that researchers do not always find specific DNA sequence variations in genes associated with muscular dystrophy (MD) is that they lack a reliable and cost-effective assay to detect point mutations in these genes. That problem may soon be solved.
Recently, Richard R. Bennett, a research technician at the Childrens Hospital, Boston, with the help of Applied Biosystems' (www.appliedbiosystems.com) VariantSEQr Resequencing System, developed a technique called Universal Conditions Direct Sequencing (UCDS).
This process of detecting DNA sequence variations in genes associated with MD uses the VariantSEQr system, an integrated system of reagents and software that allows detection of variants within previously sequenced genes in the human genome (Figure), to assist with PCR primer design for primers to be used in reactions in which researchers amplify target DNA sequences using the same thermal-cycling conditions for all primers.
The VariantSEQr system and other Applied Biosystems products are for research use only and not for use in diagnostic procedures.
Potentially, the UCDS technique will simplify the task and reduce the cost of finding point mutations present in genes associated with all different forms of MD.
Duchenne Muscular Dystrophy (DMD) has been reported to affect about 1 in 3,500 male births. The disease results from mutations in the dystrophin gene, which is located on the X-chromosome. Becker muscular dystrophy (BMD), a milder but still debilitating form of muscular dystrophy also results from mutations in the dystrophin gene. Together, DMD and BMD account for about 90% of all muscular dystrophies that occur.
In 1992, Bennett was hired by Louis Kunkel, Ph.D., associate director, professor of pediatrics, Harvard Medical School/ Childrens Hospital, department of genetics. Shortly after joining the lab, Bennett's background in computer engineering became an asset to the lab. He transformed the labs sequencing core facility into profitable sequencing and genotyping centers, directing sequencing projects for researchers in other labs.
Detecting Gene Variants
For about 15 years, a test has been available to detect the molecular basis of DMD and BMD using a multiplex PCR test, developed by Alan Beggs and Jeff Chamberlain, called the DMD/BMD multiplex PCR deletion test, which detects large deletions in the dystrophin gene.
Still in use today, this test has been an effective tool as about 60% of individuals with Duchennes have large deletions— one exon or more—in either of two different regions of the dystrophin gene. For that reason, Beggs and Chamberlain designed PCR assays for just those exons in these two hot spot regions. The other 40% are identified after a muscle biopsy followed by a staining reaction to detect for the presence of dystrophin. A negative test indicates a high probability of DMD.
However, the DMD/BMD deletion test will identify only 60% of the variant dystrophin gene sequences that cause either DMD or BMD. The other 40% of gene mutations responsible for the disease are either large duplications of one exon or more (about 5-10%) or point mutations (about 30-35%).
Although a number of different lab techniques for detecting point mutations have long existed, because of the large size of the dystrophin gene and the cost of performing these procedures, such techniques are not routinely used methods of molecular diagnosis.
A low-cost procedure for the molecular identification of BMD and DMD would require developing a reliable, cost-effective process of identifying point mutations, scattered throughout the 79 exons of the dystrophin gene. Such a procedure would provide a genotype of the dystrophin gene for over 90% of all DMD and BMD incidents.
DHPLC Technique Finds Point Mutations
In 2001, Bennett proposed sequencing all 79 exons of the dystrophin gene, and along with Dr. Kunkel and others, he subsequently published a paper in BioMed Central describing an automated method of finding point mutations in the dystrophin gene. The method involves the use of denaturing high-performance liquid chromatography (DHPLC) followed by direct sequencing.
As Bennett notes, direct sequencing offers a more definitive approach for identifying DMD. “If you sequence the DNA and find a mutation in the dystrophin gene, you have solid evidence not only of Duchennes condition but also what particular mutation caused it.“
After publishing the DHPLC method, Dr. Kunkel and Bennett still sought to find a simpler, more cost-effective method for detecting sequence variations in MD disease genes and expanding the number of genes to include all known causative genes for any type of MD or myopathy. They had a goal of using computer-aided tools to design primers for PCR assays, where using one thermal cycler, they could place all DNA samples in a single reaction plate and using the same temperature profile for all samples, perform the PCR amplification reactions necessary for direct sequencing.
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.