ZyGEM reagents are made from the highest standard reagents and tubes and dispensed in hoods within a positive pressure HEPA filtered facility. All tubes are then further treated using a proprietary technique where any remaining nucleic acids and proteins (nucleases) are destroyed in the sealed tubes. The tubes of enzyme and buffers remain unopened until they arrive at your laboratory. In addition, we perform rigorously QC testing on randomly selected tubes from every batch. The QC tests and standards are listed below. 

Waikato Innovation Park

Waikato Innovation Park - home of ZyGEM NZ

 

 

ZyGEM's markets include:

 

   •   Forensics
   •   Human Diagnostics
   •   Environmental/Biosecurity
   •   Plant (coming soon)
   •   Food Safety Testing
   •   Life Science Research
   •   Veterinary

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.

 

 

1. Production.

 

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

All kits

Activity 

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. 

DNAse Activity

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.

Efficacy

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

RNAse Activity

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.

 

EA1 model

 EA1 proteinase - the basis of the ZyGEM Nucleic extraction kits


Maldi

Main Peak: 34,600 Da (single charge). Second peak: 17,300 Da (double charge). Also shown, SDS silver-stain PAGE gel

 

RNAGEM aggressively removes RNAse A from solutions. The assay above is a real-time activity measure based on the hydrolysis of a double-fluorophore labelled riboligonucleotide. The top panel show the degradation of the oligo in the presence of a serial dilution of RNAse A. This assay is the basis of our QC test for RNAse activity. The bottom panel shows the same serial dilution, but the RNAse A has been incubated in the presence of RNAGEM. These results demonstrate how RNAGEM, rapidly degrades a notably resilient RNAse making it ideal for the lysis of cells known to be problematic for RNAse release.