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DNA Extractor Kit Application Data
Spike and recovery test of CHO cell-derived DNA
Examined the recovery rate of CHO cell-derived DNA from spiked culture supernatant to evaluate the extraction efficiency.
Methods
- Added 10 fg to 1 ng of CHO cell-derived DNA to culture supernatant of PANC-1 cells.
- Extracted DNA from the supernatant with this kit following the manual.
- Protocol: Protocol #2
- Pretreatment: None
- Conducted qPCR and measured the Cq value of the extracted DNA. The same measurement was conducted for purified water spiked with CHO cell-derived DNA without DNA extraction, this was used as standard condition.
- Create a standard curve using the mean Cq values of the standard condition, and calculated the recovery rate.
Samples
- Purified water containing CHO cell-derived DNA (no DNA extraction): Standard conditions
- DNA extracted from the culture supernatants of PANC-1 cells containing CHO cell-derived DNA by this kit: Culture supernatants
qPCR reagents
- GeneAce SYBR qPCR Mix α No ROX (Nippon Gene#319-07703)
- Optical Flat 8–Cap Strips for 0.2 mL tube strips/Plates (BIO-RAD#TCS0803)
- Hard-Shell Thin-wall 96-well PCR plates (BIO-RAD#HSP9601)
Results
| 1. Standard conditions | 2. Culture supernatants | |||||
|---|---|---|---|---|---|---|
| Spike DNA (fg) | Cq 1 | Cq 2 | Mean | Cq 1 | Cq 2 | Mean |
| 0 | ND | |||||
| 10 | ||||||
| 100 | 36.89 | ND | 36.89 | 37.44 | 36.73 | 37.09 |
| 1,000 | 33.44 | 33.91 | 33.68 | 33.94 | 34.09 | 34.01 |
| 10,000 | 30.23 | 30.17 | 30.20 | 30.72 | 30.96 | 30.84 |
| 100,000 | 26.74 | 26.68 | 26.71 | 26.90 | 27.03 | 26.96 |
| 1,000,000 | 23.39 | 23.28 | 23.33 | 23.61 | 23.48 | 23.54 |
ND: Not detected
Good extraction efficiency of CHO-derived DNA from culture supernatant within the range of 100 fg to 1 ng.
Spike and recovery test of E. coli-derived DNA
Examined the recovery rate of E. coli-derived DNA from spiked sample to evaluate the extraction efficiency.
Methods
- Added 10 fg to 1 ng of E.coli-derived DNA to purified water.
- Extracted DNA from the spiked sample with this kit following the manual.
- Protocol: Protocol #1
- Conducted qPCR and measured the Cq value of the extracted DNA. The same measurement was conducted for the spiked sample before DNA extraction.
- Create a standard curve using the mean Cq values of the spiked samples before extraction, and calculated the recovery rate after the extraction.
Samples
Purified water spiked with E. coli-derived DNA
qPCR reagents
- GeneAce SYBR qPCR Mix α No ROX (Nippon Gene#319-07703)
- COptical Flat 8–Cap Strips for 0.2 mL tube strips/Plates (BIO-RAD#TCS0803)
- CHard-Shell thin-wall 96-well PCR plates (BIO-RAD#HSP9601)
Results
Good extraction efficiency of E.Coli-derived DNA within the range of 1 pg to 1 ng.
Spike and recovery test from Human serum sample
λ/HindⅢ 10 to 1,000 pg was labeled with 32P and added to 100 μL of human serum. DNA was extracted using this kit and the recovery rate was calculated.
Reference: Ishizawa, M. et al., Nucleic Acids Res., 19, 5792 (1991).
Good extraction efficiency from serum sample.
Detection of virus DNA from human serum by PCR
Serum containing hepatitis B virus (HBV) was diluted in stages. DNA was extracted using this kit and the phenol method. HBV-DNA was amplified with PCR, and DNA was detected using agarose gel electrophoresis.
HBV-DNA was detected with higher specificity and sensitivity using this kit than phenol method.
Lane 1: Marker DNA(φⅩ174 DNA/HaeⅢ)
Evaluation For Antibody Drug
DNA extraction from high concentration protein-containing solution
Methods
- Added CHO cell-derived DNA (final DNA concentration: 2 pg/mL) to 10 – 100 mg/mL of IgG solution.
- Extracted DNA from the spiked IgG solution with this kit following the manual.
- Protocol: Protocol #2
- Pretreatment: Protocol for the sample containing more than 2 mg/mL of protein (Proteinase K treatment)
- Measured the recovery rate by qPCR
Results
Cq value of IgG solution at various concentration
| Sample | Input | Protein Concentration (mg/mL) | |||||
|---|---|---|---|---|---|---|---|
| 10 | 20 | 40 | 60 | 80 | 100 | ||
| Cq | 31.596 | 31.566 | 31.351 | 31.726 | 31.502 | 31.282 | 31.658 |
| 31.599 | 31.608 | 31.774 | 31.787 | － | 31.485 | 31.660 | |
| 31.338 | 31.861 | 31.500 | 31.850 | 31.560 | 31.369 | － | |
| Average Cq | 31.511 | 31.678 | 31.542 | 31.788 | 31.531 | 31.379 | 31.659 |
| Difference of Cq | 0 | -0.167 | 0.136 | -0.246 | 0.257 | 0.152 | -0.280 |
| Power | 1 | 0.891 | 1.099 | 0.843 | 1.195 | 1.111 | 0.824 |
| Recovery Rate (%) | 100 | 89.1 | 109.9 | 84.3 | 119.5 | 111.1 | 82.4 |
Good DNA extraction efficiency from solution containing high concentration protein (100 mg/mL).
Recovery rate of CHO cell-derived DNA from IgG solution
DNA extraction from antibody drug-mimic solution
Conducted DNA spike and recovery test from IgG solutions made of various types of buffers generally used in antibody drug and from IgG solutions containing various additives used in antibody drug.
Methods
- Made antibody drug-mimic solutions by adding 20 mg/mL of IgG to various buffers generally used in antibody drug (Table 1. ①-⑥) and to phosphate buffer containing various additives (Table 2. ①-⑩). Then add CHO cell-derived DNA (final DNA concentration: 20 pg/mL) to those antibody drug-mimic solutions.
- Extracted DNA from the spiked samples with this kit following the manual.
- Protocol: Protocol #2
- Pretreatment: Protocol for the sample containing more than 2 mg/mL of protein (Proteinase K treatment)
- Measured the recovery rate by qPCR.
Table 1. Buffer Composition
| No. | Buffer Composition |
|---|---|
| ① | 10mM PB pH6.0, 0.15M NaCl, 2mM EDTA |
| ② | 10mM PB pH7.4, 0.15M NaCl, 2mM EDTA |
| ③ | 50mM NaOAc pH5.2, 0.15M NaCl, 2mM EDTA |
| ④ | 10mM PB pH6.0, 2mM EDTA |
| ⑤ | 10mM PB pH7.4, 2mM EDTA |
| ⑥ | 50mM NaOAc pH5.2, 2mM EDTA |
Table 2. Additive-containing Buffer
Buffer: 10mM PB pH6.0, 0.15M NaCl, 2mM EDTA
| No. | Additive & Concentration |
|---|---|
| ① | Sucrose (10w/v%) |
| ② | Trehalose Dihydrate (10w/v%) |
| ③ | D(-)-Mannitol (10w/v%) |
| ④ | D(-)-Sorbitol (10w/v%) |
| ⑤ | D(+)-Maltose Monohydrate (10w/v%) |
| ⑥ | L(+)-Arginine Hydrochloride (1w/v%) |
| ⑦ | L‐Histidine (1w/v%) |
| ⑧ | Glycine (1w/v%) |
| ⑨ | Polyoxyethylene(20) Sorbitan Monooleate (Tween80) (0.5v/v%) |
| ⑩ | Polyoxyethylene(20) Sorbitan Monolaurate (Tween20) (0.5v/v%) |
Results
Cq values of each buffer
| Sample | Input | Buffer | |||||
|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | ||
| Cq | 31.473 | 31.693 | 31.544 | 31.218 | 31.058 | 31.595 | 31.181 |
| 31.364 | 31.196 | 30.973 | 31.568 | 31.085 | 31.343 | 31.112 | |
| 31.045 | 31.530 | 31.787 | 31.315 | 30.942 | 31.452 | 31.558 | |
| Average Cq | 31.294 | 31.473 | 31.435 | 31.367 | 31.028 | 31.463 | 31.284 |
| Difference of Cq | 0 | -0.179 | -0.141 | -0.073 | 0.266 | -0.169 | 0.010 |
| Power | 1 | 0.883 | 0.907 | 0.951 | 1.203 | 0.890 | 1.007 |
| Recovery Rate (%) | 100 | 88.3 | 90.7 | 95.1 | 120.3 | 89.0 | 100.7 |
DNA Recovery Rate from each buffer
Cq values of each additive-containing buffer
| Sample | Input | Additive-containing buffer | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|
| ① | ② | ③ | ④ | ⑤ | ⑥ | ⑦ | ⑧ | ⑨ | ⑩ | ||
| Cq | 31.473 | 31.826 | 31.327 | 30.907 | 31.143 | 31.071 | 31.525 | － | 31.525 | 31.013 | 31.086 |
| 31.364 | 31.194 | 30.991 | 31.759 | 31.103 | 31.279 | 31.068 | 31.442 | 31.203 | 31.340 | 30.870 | |
| 31.045 | 30.703 | － | 31.009 | － | 30.978 | 31.225 | 31.479 | 30.757 | 31.020 | 31.020 | |
| Average Cq | 31.294 | 31.241 | 31.159 | 31.225 | 31.123 | 31.109 | 31.273 | 31.461 | 31.162 | 31.124 | 30.992 |
| Difference of Cq | 0 | 0.053 | 0.135 | 0.069 | 0.171 | 0.185 | 0.021 | -0.167 | 0.132 | 0.170 | 0.302 |
| Power | 1 | 1.037 | 1.098 | 1.049 | 1.126 | 1.137 | 1.015 | 0.891 | 1.096 | 1.125 | 1.233 |
| Recovery Rate (%) | 100 | 103.7 | 109.8 | 104.9 | 112.6 | 113.7 | 101.5 | 89.1 | 109.6 | 112.5 | 123.3 |
DNA Recovery Rate from each additive-containing buffer
Good DNA extraction efficiency from various buffer and additive.
