Brief summary of prepgem and forensicgem Properties. ZyGEM: Sept

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1 Brief summary of prepgem and forensicgem Properties ZyGEM: Sept

2 SUMMARY OF forensicgem AND prepgem PROPERTIES Enzyme Description and purity The key component of forensicgem and prepgem is a neutral proteinase from the Bacillus sp. Strain EA1. This enzyme was selected from a panel of over 80 proteinases studied for its efficacy in degrading animal tissue in buffer conditions compatible with the PCR. The key difference between forensicgem and prepgem is that forensicgem is formulated and validated for forensic samples. This proteinase is well-researched having studied by more than 10 people for over 20 years, with at least 19 different publications describing it. Organism Figure 1. Mt Erebus, Antarctica the source of Bacillus sp. EA1. Bacillus sp. EA1 is a thermophilic member of the Bacillus caldolyticus group of bacteria. The organism was isolated from a volcanic vent close to the peak of Mount Erebus Antarctica (Figure 1). The phylogeny based on 16S rrna sequence data is shown in Figure 2. Figure 2. Consensus phylogenetic tree constructed from the six most parsimonious (MP) trees. Scale bar represents and expected nucleotide substitution rate of 0.01 changes per site. Values on branches indicate percent support derived from 100 boot-strapped trees. Enzyme production The enzyme is produced in recombinant form from strains of Escherichia coli K12 using a public domain expression system Figure 3. Mass spectroscopy trace and a silver-stained polyacrylamide gel of the purified enzyme.

3 The enzyme is QC tested for the following: 1. Activity on azocasein. 2. The presence of bacterial DNA. 3. The presence of human DNA. 4. The presence of mtdna (HV1). 5. Contamination by single- and doublestranded deoxyribonucleases 6. RNAse activity. 7. Identifiler profile on Buccal swabs and Blood extraqcts (forensicgem only). 8. Identifiler profile on enzyme (forensicgem only). 9. Efficacy on extraction from blood (PCR of low copy number nuclear gene (GAPDH; prepgem only). An example of one test in the standard QC is shown below. Figure 2. The rates of activity of serial dilutions of RNAse A on a dual-labelled RNA. This is compared to the RNAse activity of 1U of forensicgem Cleavage of an oligo dual-labelled ribonucleotide separates FAM and TAMRA labels thereby increasing fluorescence. Y axis = relative fluorescence units. X Axis = number of 30 second cycles, Z axis = number of Kunitz Units of RNAse A* The results demonstrate that the forensicgem enzyme contains no detectable RNAse activity on this substrate. *One Kunitz units is defined as the amount of activity which is capable of causing within 1 minute a decrease in absorbance at 300 nm equivalent to the maximum possible change in a 0.05 % solution of yeast RNA at 25 C, ph 5.0 (Kunitz, M.A., A spectrophotometric method for the measurement of ribonuclease activity, J. Biol. Chem., 164, , 1946). Molecular Data Enzyme The proteinase from Bacillus sp. EA1 is a neutral proteinase from the thermolysin family. It is presently the most thermostable member of this family reported. The proteinase is most closely related to a proteinase from Bacillus caldolyticus YP-T but has a T opt of 10 C high than this organism. Desirable characteristics of this enzyme for the applications developed by ZyGEM are: 1. Thermal properties a. EA1 proteinase functions optimally at 75 C with the advantages this higher temperature DNA extraction as listed in the patent application. b. EA1 proteinase is easily heat -inactivated without the addition of chelating agents. c. EA1 proteinase has greatly reduced activity at temperatures less that 40 C. This property makes it unsuitable for use in combination with mesophilic enzymes. d. EA1 proteinase has minimal activity at room temperature allowing easy storage, ambient temperature delivery and an enhanced shelf life. No other known proteinase has a temperature-activity profile as suitable as EA1 proteinase at these four temperatures. The nearest relative to EA1 proteinase (on the basis of amino acid sequence) is the neutral proteinase from Bacillus caldolyticus. This enzyme has a lower optimal temperature and significant residual activity at lower temperatures. It therefore only fulfils three of the four pre-requisites. 2. Any enzyme used for DNA applications must work in buffer conditions suitable for the PCR (i) neutral (ii) low ionic strength (iii) activity in buffers with minimal Ca 2+. These properties rule out most enzymes described elsewhere. 3. Broad specificity and high specific activity. 4. Ease of production. EA1 proteinase can be made in a recombinant host with at mesophilic temperatures. Enzymes with lower T opts tend to be toxic to recombinant hosts and others form inclusion bodies

4 Enzyme Properties ph profile 4 Activity [%] ph Figure 5. ph profile of prepgem Stability data prepgem is stabilized by Ca 2+. In presence of 5 mm CaCl 2 75 C no loss of activity after 7 days; t 1/2 = 1h at 85 C t 1/2 = 60 min (10 mm CaCl2 t 1/2 = 115 min) 95 C t 1/2 = 10min However, Ca2+ is not added to the storage buffer because this divalent ions are undesirable in the PCR. As a consequence, the proteinase as prepared by ZyGEM has a reduced stability but is still sufficiently robust for easy handling and transportation. In the storage buffer (with 20% Glycerol) there is only a 10% loss of activity after 100 days at room temperature. In the standard reaction buffer (without glycerol) there, prepgem loses only 20% activity after a year at room temperature (Figure 6). [A] No glycerol Storage of EA1 proteinase (1000 U/ml) 5 mm HEPES, 0.01% Triton X-100, 1mM MgCl2, ph 7.0 [B] 20% glycerol Storage of EA1 proteinase (1000 U/ml) 5 mm HEPES, 0.01% Triton X-100, 1mM MgCl2, ph 7.0, 20% Glycerol. Figure 6. Rate of activity loss of prepgem in the storage buffer with and without Glycerol.

5 Stability under laboratory abuse Typically, tubes of enzymes are repeatedly removed from storage at -20 C and transferred either to ice or to room temperature. Figure 7 shows the loss of activity of prepgem over 30 daily cycles of freeze-thaw. Negligible loss can be seen when the temperature shift is just above freezing point and a loss of approximately 15% is seen when the enzyme is transferred from the freezer to room temperature 30 times. Response to inhibitors Figure 7. Rate of activity loss of prepgem under a variety of freeze-thaw cycles. The following data were generated using Azocasein as substrate and 0.02% inhibitor Inhibitor % Inhibited Gramicidin S Trypsin (soy bean) 9.05 Trypsin (egg) 5.97 Gramicidin 3.90 Trypsin (lima bean) 2.26 Bacitran 0.82 Benzamidine 0.00 Control 0.00 Comparative specific activitiy with proteinase K 10 minute assay 100 minute assay Figure 8. Specific proteolytic rates of proteinase K and prepgem at different temperatures. The substrate used was 0.2% BSA. Values are shown relative to the specific activity of prepgem at 75 C Relative activity of prepgem on various substrates [6] Azocasein 1.00 Azocollagen 1.37 Keratin 0.02 Hide powder azure 1.12 Elastin congo red 0.00 Casein 2.46 Collagen 0.26 Haemoglobin 0.76 Bovine serum albumin 0.37 Ovalbumin 0.47 Lactalbumin 0.19 FLAGA 0.00 Suc(ala) 3pNA 0.00

6 Effect of additives on prepgem activity. Activity loss in the presence of detergents and typical reducing agents shows that EA1 is robust enough to withstand a variety of denaturants. Several of these denaturants can be used at concentrations sufficiently high for them to be used as an adjuvant for tissue lysis (Figures 9 and 10) or may be contaqminants in the tissue or reagents (Figure 11).. cd Figure 9. Residual activity of prepgem in the presence of reducing agents (10 minute incubation). Figure 10. Residual activity of EA1 in the presence of detergents. Both Azocasein and BSA assays were [A] Formalin [B] Glycerol Activity [%] 0% glycerol % glycerol 75 20% glycerol 41 30% glycerol 23 Figure 11.Relative activities of prepgem in the presence of formalin and glycerol

7 DNA yields Liquid Blood The following yields were calculated for forensicgem using both a blot-based system, ABI Quantiblot and a qpcr method, Quantifiler. Sample size = 2.5 µl Blood Final Volume forensicgem extraction = 100 µl Quantiblot Quantifiler TOTAL YIELD: ng per µl blood 25.2 ± ± 22.8 CONC. in extract ng /µl 0.6 ± ± 0.46 QIAGEN report typical yields of 3 µg of DNA from 2 ml of blood (2.5 x 10 5 Leukocytes per ml) for their QIAamp columns. This equates to a yield of 1.5 ng DNA / µl of blood. Chemagen report yields of µg of DNA from 3 ml of blood. This equates to a total yield of ng DNA / µl of blood. Blue: Examples of Quantifiler traces from forensicgem blood extracts. Samples from four individuals were used. Red: standards generated using the manufacture's DNA Yields from blood samples (four individuals) using forensicgem and other extraction methods. Error bars are one standard deviation. Volume of blood used: forensicgem 2.5 Chemagic 50 QIAamp 5 Phenol 5 Final Volume of extract: forensicgem 100 MagBead 100 QIAGEN 50 Phenol 50 Note: Different blood samples were used to create this data and the quantification table above. Variation is typical between blood samples due to different titres of white blood cells. Notes: 1. The method used for forensicgem was as follows: a. The following extraction mixture was made: i. 2.5 µl of fresh blood ii. 97 µl of Buffer 3. iii. 1 µl of forensicgem TM b. Samples were incubated at 75 C for 15 minutes and 95 C for 5-15 minutes. c. The extracts were centrifuged at 13,000 x g for 5 minutes. 2. The phenol method used 20 µl of blood treated with proteinase K in a 200 µl volume. Samples were extracted with phenol, phenol+chloroform+iaa (25:24:1) and chloroform. Samples were precipitated in ethanol and then resuspended on 50 µl of TE buffer. 3. All other methods were scaled variants of the manufacture's instructions.

8 Buccal Swabs. The following yields were calculated for forensicgem using both a blot-based system, ABI Quantiblot and a qpcr method, Quantifiler. Sample size = ¼ of a swab Final Volume forensicgem extraction = 100 µl Quantiblot Quantifiler TOTAL YIELD: ng per µl ¼ swab 175 ± ± 88.9 CONC. in extract ng /µl 1.8 ± ± 0.89 On this basis a complete swab would be expected to yield ~ 940 ng of DNA. Large variation in yields is common with buccal swabs samples would be expected to vary from between 500 and 2000 ng. QIAGEN reports similar yields for the QIAamp 96 DNA Swab BioRobot Procedure. Blue: Examples of Quantifiler traces from forensicgem buccal swab extracts. Four extractions are shown. Red: Standards generated using the manufacture's DNA Yields from buccal swabs (four individuals) using forensicgem and other extraction methods. Error bars are one standard deviation. Preparation of Swab samples. Individual swabs were washed in 400 µl of buffer. This suspension was used the four extraction methods. Volume of suspension used: forensicgem 100 MagBead 100 QIAGEN 100 Phenol 100 Final Volume of extract: forensicgem 100 MagBead 100 QIAGEN 50 Phenol 50 Note: Different Buccal swabs were used to create this data and the quantification table above. Buccal swabs vary greatly from sample to sample. Notes: 1. The method used for forensicgem was as follows: a. The following extraction mixture was made: i. 100 µl of swab suspension ii. 2 µl of 50 x Buffer 3H. iii. 1 µl of forensicgem TM b. Samples were incubated at 75 C for 15 minutes and 95 C for 5-15 minutes. 2. All other methods were scaled variants of the manufacture's instructions.

9 References. [1] Collet, C., University of Waikato Thesis: Proteinases Suitable for use in Molecular Biology. (2001). [2] Toogood, H. S., University of Waikato Thesis: Thermostable Proteinases from Thermophilic Microorganisms. (1998) [3] Coolbear, T., Monk, C., Peek, K., Daniel, R.M. and Morgan, H.W., Laboratory-scale investigations into the use of extreme thermophilic proteinases for cleaning ultrafiltration membranes fouled during whey processing. Journal of Membrane Science 67 (1992) [4] Wilson, S.A., Young, O., Coolbear, T., Daniel, R.M., The use of proteinases from extreme thermophiles for meat tenderizing. Meat Science 32 (1992) [5] Coolbear, T., Whittaker, J.M., Morgan, H.W. and Daniel, R.M., The effect of metal ions on the activity and thermostability of the extracellular proteinase from Bacillus strain EA.1. Biochemical Journal 286 (1992) [6] Coolbear, T., Eames, C.W., Casey, Y., Daniel, R.M. and Morgan, H.W., Screening of strains identified as extremely thermophilic bacilli for extracellular proteolytic activity and general properties of the proteinases from two of the strains. Journal of Applied Bacteriology 71 (1991) [7] Beynon, R. J., Bond, J. S., Proteolytic enzymes: a practical approach. IRL press, Oxford. (1989). [8] Bundschuh, A., University of Waikato: Laboratory book data. (2004). [9] Cowan, D.A. and Daniel, R.M., Rapid purification of two thermophilic proteinases using dye-ligand chromatography. Biochemical and Biophysical Methods, 32 (1996) 1-6.