Detection of rare mutant alleles by restriction endonuclease-mediated selective-PCR: Assay design and optimization

Background: Restriction endonuclease-mediated selective (REMS)-PCR, allows detection of point mutations, deletions, and insertions. Reactions require concurrent activity of a restriction endonuclease (RE) and a DNA polymerase, both of which must be sufficiently thermostable to retain activity during thermocycling. The inclusion of the RE in REMS-PCR inhibits amplification of sequences containing the RE recognition site, thus producing selective amplification of sequences that lack the RE site. Methods: Assays were used that allowed the selection of conditions that produce concurrent RE/DNA polymerase activity. The RE thermostability assay involved thermocycling a RE under various conditions and assessing residual cleavage activity at various time points. Conditions found to preserve RE activity during thermocyling were then tested for their compatibility with DNA polymerase-mediated PCR. Results: A range of conditions that preserve activity of the RE BstNI over 30 cycles of PCR was identified. A subset of these conditions was subsequently found to mediate specific amplification using Taq DNA polymerase. These conditions were used to develop a REMS-PCR protocol for the detection of mutations at codon 12 of the K-ras gene. This protocol allowed the detection of 1 mutant allele in a background of 1000 wild-type alleles. The presence of primer sets for RE and PCR control amplicons provided unambiguous assessment of mutant status. Conclusion: Implementation of the assays described may facilitate development of REMS-PCR assays targeted to other loci associated with disease. (C) 2000 American Association for Clinical Chemistry

Enzyme activity in the aestivating Green-striped burrowing frog (Cyclorana alboguttata)

Green-striped burrowing frogs (Cyclorana alboguttata) can depress their resting metabolism by more than 80% during aestivation. Previous studies have shown that this species is able to withstand long periods of immobilisation during aestivation while apparently maintaining whole muscle mass and contractile performance. The aim of this study was to determine the effect of prolonged aestivation on the levels of metabolic enzymes (CCO, LDH and CS) in functionally distinct skeletal muscles (cruralis, gastrocnemius, sartorius, iliofibularis and rectus abdominus) and liver of C. alboguttata. CS activity was significantly reduced in all tissues except for the cruralis, gastrocnemius and the liver. LDH activity was significantly reduced in the sartorius and rectus abdominus, but remained at control (active) levels in the other tissues. CCO activity was significantly reduced in the gastrocnemius and rectus abdominus, and unchanged in the remaining tissues. Muscle protein was significantly reduced in the sartorius and iliofibularis during aestivation, and unchanged in the remaining muscles. The results suggest that the energy pathways involved in the production and consumption of ATP are remodelled during prolonged aestivation but selective. Remodelling and subsequent down-regulation of metabolic activity seem to target the smaller non-jumping muscles, while the jumping muscles retain enzyme activities at control levels during aestivation. These results suggest a mechanism by which aestivating C. alboguttata are able to maintain metabolic depression while ensuring that the functional capacity of critical muscles is not compromised upon emergence from aestivation. © 2010 Springer-Verla