MLX BCG Buccal Cell Genomic DNA Extraction Kit. Performance Characteristics

MLX BCG Buccal Cell Genomic DNA Extraction Kit Performance Characteristics Monolythix, Inc. 4720 Calle Carga Camarillo, CA 93012 Tel: (805) 484-8478 monolythix.com

of 9 MLX BCG Buccal Cell Genomic DNA Extraction Kit Performance Characteristics The Monolythix MLX-BCG buccal cell genomic DNA extraction kit is a convenient high throughput extraction system that can be used with manual pipettes or automated liquid handling systems. The current kit includes processes for moving liquid though the monolith and elution from the monolith using a microplate centrifuge. The kit consists of a set of plates and reagents required for lysis, binding, wash and elution of purified buccal cell DNA. Unlike many other DNA preparation systems, chaotropic salts are not used. The kit is compatible with buccal cells preserved in the MLX-BCG Collection Media. The MLX DNA binding monolith is a proprietary sponge-like polymer with very high DNA binding capacity (See Figure 1). After proteolysis and washing, the DNA is eluted in a minimal volume of elution solution. Aliquots of the eluted DNA demonstrate no PCR inhibition in 25 ul real time PCR assays. In this note, we demonstrate the performance of the system on different buccal samples. Figure 1. MLX DNA binding matrix.

of 9 Cell Collection Cell collection is performed by scraping the inner surface of one cheek with one swab and eluting the intact cells into 500 ul of MLX-BCG Collection Media. Only 50 ul of this material is needed for each monolith well. For process validation, cells may be counted by first diluting the cells 1:4 in 10mM Tris ph 7.5, diluting this mixture 1:1 with a 0.05 wt. % Methylene blue solution in water (e.g. Sigma Catalog number 319112), and applying the cells to a hemocytometer. Figure 2. Aggregated buccal epithelial cells stained with methylene blue. Due to some cell overlap, each dark staining nucleus must be counted as a cell. Buccal scrapes often contain a variable number of small nucleated white blood cells. These nuclei contribute the same amount of DNA as each larger epithelial cell nuclei. If a test sample contains a large fraction of white blood cells, count these cells when calculating the total cell count used for extraction.

of 9 Cell Counts Unlysed buccal cells are stable in the preservative for ~ 7 days at room temperature and an additional 14 days at 4 C. Collected cells should not be stored for more than 3 weeks. Cells will settle with storage. Due to settling, it is imperative that buccal cells in collection media are vortexed before aliquots are taken. If the collection media with cells is not cloudy upon vortexing, there may not be enough cells to obtain standard recoveries with the extraction kit. The MLX-BCG monolith matrix has been optimized to extract high fractions of available DNA from buccal cells. Buccal cell counts ranging from ~ 30,000 60,000 per 50 ul (600,000 to 1,200,000 cells per ml) are typical, and the kit is optimized to work with cell counts in this range. If too many cells are used or if lysis is incomplete, the MLX-BCG matrix plugs can be overloaded, resulting in reduced flow characteristics. If overloaded, additional centrifugation time may be used to pass loading wash and elution solutions through the matrix. However, the quality of the DNA eluted from flow-restricted monolith plugs may be compromised. For greater DNA yields, the safest process is to apply collected cells to more than one well. Digestion Temperature The process of cell lysis and protein digestion appears to be optimal in the 50-55 C range (see Figure 3). Some protocols recommend a post lysis heat treatment to remove residual protease activity. In our experience, heating to >90 C after digestion decreases DNA recovery significantly, and is therefore discouraged. In our assays, no evidence of residual protease activity in real time PCR assays is observed, suggesting that all protease is removed by the washing steps in the standard protocol. Digestion Time Digestion of cellular and nuclear proteins appears to be sufficient in as little as 5 minutes when performed in a thermal cycler in a volume of 200uL (Figure 4). Other heating devices may be used, but care should be taken to ensure adequate warming of the lysis solution. A 10 minute digestion period is recommended, which includes a margin of safety for slow heating or higher cell counts. Elution Volumes In addition to the number of cells loaded, the volume used to elute monolith-bound DNA will determine the total recovery and DNA concentration. Between 50 ul and 150 ul, the total recovery of DNA will increase (See Table 1 and Figure 5). However, the highest concentration of DNA will be obtained in the first 50 ul. For most applications, 60 ul of elution solution is satisfactory. A 60 ul elution volume will recover between 50% and 70% of the available DNA while maintaining a relatively high concentration of > 2.0 ng/ul if sufficient cells are loaded. For greater net yields, sequential elution with smaller volumes results in higher recovery than a single large volume (e.g. 50 ul plus 50 ul instead of 100 ul).

of 9 Figure 3. Effect of lysis temperature on DNA yield. The difference in yield between 50 C and 60 C is small. The recommended lysis temperature is 55 C. Elution Volume: 60 ul. Input cell count: 50,000: Means of 8 wells. Figure 4. Fast lysis of buccal cells using MLX protease and MLX conditioner at 55 C. Input cell count: 46,000. Means of 5 wells. ng DNA Yield 350 300 250 200 150 100 50 0 DNA Eluted (ng) 252 245 258 251 0 10 20 30 40 Digestion Time (min)

of 9 Table 1. DNA recovery based on elution volume from an experiment where 39,500 cells were loaded. The first elution volume was 50 ul, with 4 subsequent 25 ul elution steps. Each eluted volume was quantitated by real time PCR. The data are cumulative - each eluted amount was added to the prior eluted amounts. Elution Volume 50 75 100 125 150 Cum. ng 116 165 194 215 227 Cum. Recovery 49% 70% 82% 91% 96% Final ng/ul 2.3 2.2 1.9 1.7 1.5 Figure 5. Cumulative DNA recovery based on elution volume. As elution volume increases, recovery approaches 100% while concentration decreases slightly. % DNA Recovery 120% 100% 80% 60% 40% 20% 0% Elution Volume vs Recovery and Concentration 50 75 100 125 150 2.3 2.2 1.9 1.7 1.5 50 75 100 125 150 Elution Volume (ul) 2.5 2.0 1.5 1.0 0.5 0.0 ng/ul Cumulative Recovery ng/ul

of 9 Reproducibility and Absence of Cross-contamination To avoid well to well cross-contamination, the MLX-BCG kit utilizes a deep well plate to recover eluted DNA. For medium term storage, the DNA should be transferred from the deep well plate to polypropylene tubes or wells that can be tightly sealed or capped and stored at 4 C. To demonstrate reproducibility, 6 swabs from each of 3 individuals were collected into six independent sample collection tubes containing MLX Collection Media. The 6 sampless from each individual were pooled. To check for cross-contamination, 150 ul of Lysis Solution containing MLX protease was pipetted into all wells of a 96 well lysis PCR style plate. Next, 50 ul aliquots of pooled cell lysate were redistributed into alternating positions of the lysis plate (Figure 6) and mixed by pipetting the 50 ul up and down 3 times. 45 of the remaining 48 wells received lysis buffer only. The final 3 wells of 48 received 3 different dilutions of commercially purified, BamHI digested human genomic DNA for quantitation. The lysis plate was sealed with plate sealing film and heated to 55 C for 10 minutes, and then cooled to room temperature. Figure 6. Positioning of buccal cell samples in the 96-well lysis plate. 48 of 96 wells were loaded with buccal cells. All but remaining wells except 3 (standards) received lysis buffer only. Donor1 Donor2 Donor3 DNA Quantitation Kit-extracted DNA was quantitated using real time PCR for human beta-actin. Quantitative standards were dilutions of commercially pure genomic DNA. The standards were freshly diluted from an ethanol precipitated stock of BamHI-digested (to improve uniformity) genomic DNA that was quantitated on a UV transparent plates using a Molecular Devices SpectraMAX 190 plate reader. The use of UV absorbance to quantitate unprecipitated MLX kit-extracted DNA is not recommended. Real time PCR, double- stranded DNA binding dye quantitation, or functional assays appropriate to the downstream application are better suited to avoid the influence of substances that may interfere with accurate UV quantitation.

of 9 Reproducibility Table 3 shows the reproducibility of extracting DNA from different individuals. The ng yields are reasonably consistent within each individual. Based on our experience with commercially purified DNA, we believe the variability observed is due variable numbers of cells applied to each well or due to variation in the sampling of large DNA fragments. Input cell counts varied from individual to individual, resulting in differing yields of DNA. However, the percent recoveries were 82%-99% based on cell counts. This demonstrates that the MONOLYTHIX buccal cell extraction system delivers a high fraction of the DNA contained in a given sample. All of the control wells filled with preservative only showed no amplification up to cycle 40, establishing the absence of well-to-well cross-contamination when the MLX provided labware and procedures are used. Figure 7. Beta actin real time PCR (log plot) of 48 positive buccal samples in a 96 well plate (quantitative standards excluded). The Cq distribution is very narrow, suggesting highly reproducible performance of the extraction system.

of 9 Table 2. Cq for 48 samples run in a checkerboard pattern in a 96-well plate. 1 2 3 4 5 6 7 8 9 10 11 12 A 22.9 No Cq 22.9 No Cq 22.6 No Cq 22.7 No Cq 23.0 No Cq 22.9 No Cq B Std. 23.0 No Cq 22.8 No Cq 22.4 No Cq 21.9 No Cq 23.1 No Cq 23.0 C 23.7 No Cq 22.8 No Cq 22.5 No Cq 22.2 No Cq 23.4 No Cq 23.3 Std. D No Cq 23.1 No Cq 23.1 No Cq 22.7 No Cq 22.6 No Cq 23.3 No Cq 23.5 E 23.1 No Cq 23.5 No Cq 22.9 No Cq 22.6 No Cq 23.2 No Cq 23.3 No Cq F No Cq 23.2 No Cq 23.0 No Cq 22.6 No Cq 22.6 No Cq 22.5 No Cq 23.5 G 23.1 No Cq 23.4 No Cq 22.4 No Cq 22.6 No Cq 23.5 No Cq 23.2 No Cq H No Cq 22.7 No Cq 23.0 Std. 22.6 No Cq 22.3 No Cq 23.3 No Cq 23.4 Table 3. DNA recovery from buccal cells from 3 individual donors with input cell counts ranging from 35,000 to 50,000 cells. Donor A Donor B Donor C Cells Loaded 41,875 50,000 35,000 Mean ng Recovered 205 296 188 Mean % Recovered 82% 99% 90% Std. Dev 13% 17% 18% Summary The MLX BCG buccal cell genomic DNA purification system is a fast and reliable kit for collection, transport, and extraction of mammalian genomic DNA for real time PCR and other applications. The kit is distributed in formats compatible with standard 96-well plates, making it suitable for high throughput manual or automated operations. Learn about other monolith matrix molecular applications at www.monolythix.com.