ZyGEM Extraction enzyme
The prepGEM, forensicGEM and RNAGEM kits are distinct formulas based on a neutral proteinase from Bacillus sp. str. EA1. The following sections outlines the QC that ALL our kit variants undergo. Below this section are descriptions of the QC tests for specific kits.
Recombinant enzyme is produced in E. coli and purified to a single MALDI-TOF peak and a single band on a silver stained gel (figure to the right). As part of the process, the purified enzyme is subjected to nuclease treatment.
2. Quality Control
Fluorometric, comparative assay to avoid substrate variation. All tubes are assayed to be within 5% of target. Enzyme is supplied 10% over activity
Absence of human gDNA
None detected. Test is sensitive to ~3 pg (1 human genome equivalent)
Absence of Bacteria DNA
None detected. Tested with universal 16S rRNA gene primers. Special care is taken with the PCR reagents that are often contaminated with trace bacterial DNA.
Absence of mtDNA
None detected. Tested with universal d-loop primers in a qPCR.
No endo- or exonuclease detected. 1µl of proteinase is incubated in the presence of 10 µM primers and 0.1 ng human DNA templates for 15 minutes at 37°C and 15 minutes at 75°C. The DNA is then amplified for 30 cycles. The sensitivity of the test determines that there is less than 0.001 U DNAse I equivalent.
Enzyme and buffers are tested against the control enzyme samples for efficacy at extracting from the appropriate substrate for the kit.
Additional QC for RNAGEM
Enzyme and buffers are tested for the presence of RNAse activity using a fluorimetric test capable of detecting as little a 10-7 Units of RNAse A
Proteinase Activity on RNAse A
The enzyme is shown to be capable of removing 0.1 U of RNAse A. RNAGEM is a powerful, broad-specificity, thermophilic proteinase that aggressively destroys ribonucleases, and no residual RNAse activity can be detected in extracts generated from human cell lines. The graphs to the right demonstrate RNAGEM's activity on RNAse A – a notably robust ribonuclease. A serial dilution of the RNAse A was made and one set of tubes treated left untreated (top) and the other set treated with RNAGEM for 5 minutes. As can be seen, all but the highest concentrations (which would never be encountered in biological samples) have been completely neutralised.
Additional QC for forensicGEM
Efficacy with profiling kits
Enzyme and buffers are tested for their ability to generate DNA suitable for Indentifiler and Powerplex profiles from saliva and blood. Substrate-free samples must generate no peaks. Validation data has been obtained with our forensicGEM kits by a number of laboratories.