Prof. Larry Wangh and his lab are interested in detecting changes in mitochondrial genomic sequences that result from aging, disease, or drugs. To do this, they use LATE-PCR, an advanced form of asymmetric PCR, to detect mutations in the mitochondria by using multiplexes to study many mitochondrial genes at the same time. LATE-PCR generates single DNA strands that are easily diluted for sequencing. In the past. they have only been able to sequence one DNA strand from these multiplex reactions.
In a recent publication in Nucleic Acid Research, staff members Yanwei Jia and John Rice, along with Molecular and Cell Biology grad student Adam Osborne, describe the development of a blocking reagent that allows them to sequence both strands of the product DNA, thus allowing for the easy verification of mutations.
The figure at right shows that without a blocker (BLK), one is not able to obtain the excess (XP) strand sequence from a multiplex reaction. Using a blocker one is able to get not only the limiting (LP) strand, but also the excess strand from the same multiplex
