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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
  1. Added 10 fg to 1 ng of CHO cell-derived DNA to culture supernatant of PANC-1 cells.
  2. Extracted DNA from the supernatant with this kit following the manual.
    1. Protocol: Protocol #2
    2. Pretreatment: None
  3. 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.
  4. Create a standard curve using the mean Cq values of the standard condition, and calculated the recovery rate.
Samples
  1. Purified water containing CHO cell-derived DNA (no DNA extraction): Standard conditions
  2. 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
  1. Added 10 fg to 1 ng of E.coli-derived DNA to purified water.
  2. Extracted DNA from the spiked sample with this kit following the manual.
    1. Protocol: Protocol #1
  3. Conducted qPCR and measured the Cq value of the extracted DNA. The same measurement was conducted for the spiked sample before DNA extraction.
  4. 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
  1. Added CHO cell-derived DNA (final DNA concentration: 2 pg/mL) to 10 – 100 mg/mL of IgG solution.
  2. Extracted DNA from the spiked IgG solution with this kit following the manual.
    1. Protocol: Protocol #2
    2. Pretreatment: Protocol for the sample containing more than 2 mg/mL of protein (Proteinase K treatment)
  3. 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
  1. 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.
  2. Extracted DNA from the spiked samples with this kit following the manual.
    1. Protocol: Protocol #2
    2. Pretreatment: Protocol for the sample containing more than 2 mg/mL of protein (Proteinase K treatment)
  3. 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.

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