DNA Extractor® Kit

Kit component

50 tests

Outline

Residual DNA testing for vaccines and biopharmaceuticals

This is sodium iodide method based DNA extraction kit to isolate residual host cell DNA from biological samples. This kit follows the residual DNA extraction protocols described in the United States Pharmacopeia (USP) 43-NF38, <509> Residual DNA Testing.
The isolated DNA can be used for qPCR. This is suitable for the residual DNA testing derived from host cells including CHO cells, E.coli and yeast.
This kit can also be used for DNA isolation for Threshold® System provided by Molecular Devices, LLC, which can quantify total DNA regardless of the origin.

Virus DNA quantification in human serum

This can be used for virus DNA isolation from human serum.

Features

• Efficient extraction of trace DNA (100-1,000 fg）
• Perform in a single tube for whole process
• Extraction complete in 60-90 minutes
• Suitable for quantification by qPCR and Threshold® System (Molecular Devices, LLC)
• Pretreatment protocol for samples containing high concentration protein

Basic operation of DNA extraction by sodium iodide method

1. Add sodium iodide, a chaotropic ion and detergents to solubilize protein and lipids.
2. Add 2-propanol and DNA is coprecipitated with glycogen.
3. Collect DNA pellets.

Application

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.
   -Protocol: Protocol #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

ND: Not detected

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.
   -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

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).

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.

Lane 1: Marker DNA(φⅩ174 DNA/HaeⅢ)

Evaluation For Antibody Drug

DNA extraction from high concentration protein-containing solution

Conducted DNA spike and recovery test from various concentration of IgG 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.
   -Protocol: Protocol #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

Recovery rate of CHO cell-derived DNA from IgG solution

Cq value of IgG solution at various concentration

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.
   -Protocol: Protocol #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

DNA Recovery Rate from each buffer

Cq values of each buffer

Cq values of each additive-containing buffer

DNA Recovery Rate from each additive-containing buffer

Overview / Applications