Rapid DNA Extraction from Dried Blood Spots

Dr Brian Douglas

Molecular Biologist, Bento Lab

Many areas of medical or animal research rely on dried blood spots (DBS) on filter paper to store DNA samples, but extracting DNA from DBS can be time-consuming or require expensive kits and reagents.

In this post we suggest four quick, easy, inexpensive, and accessible methods that could be tried to extract PCR-amplifiable DNA from DBS, based on methods reported in the published literature.

The reagents should be easy to buy and make yourself, or can be easily made by modifying our HotSHOT DNA Extraction Kit with solid sodium hydroxide (NaOH) and sterile distilled water.

Method 1: 98 °C Incubation in Phosphate-Buffered Saline, Centrifugation, and Direct PCR (Aslam et al., 2023).

This method was described by Alsam et al. (2023) for use for PCR-based sexing of chickens. It involves extracting DNA from dried blood spots via near-boiling in a pH-neutral buffer (phosphate-buffered saline or PBS), and is therefore probably the easiest, cheapest, and safest method that we’ve seen for extracting amplifiable DNA from dried blood spots. It can also be used for DNA extraction from feathers, and it may have other applications as a quick simple extraction method.

Alsam et al. (2023) reported successful PCRs of amplicons up to 500 bp from dried blood spots and feather samples from chickens of up to 5 years of storage (longer storage durations were not tested).

Protocol:

  1. Add between 1/6  to 1/2 of a 6 mm diameter dried blood spot to a 200 µL PCR tube.
  2. Add 50 µL of 1x PBS buffer
  3. Incubate at 98 °C for 10 minutes
  4. Centrifuge rapidly for 10 seconds
  5. Use 1– 2 µL of crude DNA extract for PCR

Health and Safety Precautions:

No hazardous reagents used. Wear gloves and eye protection as a standard laboratory precaution.

Reagents needed:

1x PBS Buffer (NaCl: 137 mM; KCl: 2.7 mM; Na2HPO4: 10 mM; KH2PO4: 1.8 mM; pH: 7.4).

Advantages:

  • Rapid: Extractions involve a 10-minute incubation and a 10 second spin.
  • Cheap: PBS buffer is inexpensive and 1 L would allow 20K x 50 µL extractions.
  • Safe: PBS is pH neutral and a 1x solution has no risks to health
  • Simple: A two-step process (incubation and centrifugation)
  • Produces an extract stock: extracted DNA can be used for multiple PCRs or stored as a solution for future use.

Disadvantages:

  • It’s a recent approach and has not been independently verified in other studies or verified with blood from animals other than chickens.
  • The 10 second spin requires a centrifuge.
  • Produces single-stranded DNA which is less stable than double-stranded DNA.
  • The extraction is very likely to fragment DNA.
  • Shelf-life of PBS-extracted DNA has not been investigated.

Possible issues:

  • This method was shown to work with dried blood spots from chickens, but bird blood is nucleated and contains DNA in amounts several orders of magnitude larger than that of mammal blood. This method should be tested and optimised if it is to be used with non-nucleated blood from other animals or humans.

Protocol Reference:

Aslam et al. (2023). Effect of storage temperature and duration on direct PCR amplification of various feather types and DBS matrices. Gene, 854, 147116.

Method 2: NaOH Lysis, Neutralisation Wash, and Direct PCR from DBS Discs (Zhou et al., 2006)

This method was developed by Zhou et al. (2006) for use with dried blood spots from sheep. It uses a two-step weak NaOH lysis, washing in a Tris-HCl buffer neutralisation buffer, followed by drying the blood spot disc and using it for direct PCR.

Protocol:

  1. Place a 1.2 mm diameter blood spot in a 200 µL PCR tube and add 200 µL of 20 mM NaOH.
  2. Incubate the tube for 30 mins at room temperature for fresh blood spots, or at 50 °C for older blood spots.
  3. Remove the lysis buffer and add 200 µL of low EDTA TE buffer (10 mM Tris-HCl pH 8, 0.1 mM EDTA) and mix well with inversions for 2 mins to wash the blood spot.
  4. Remove the neutralisation buffer, and leave the disc to air dry.
  5. Add the disc to 20 µL of PCR mix for direct PCR.

Health and Safety Precautions:

No hazardous reagents used. Avoid contact with eyes. Wear gloves and eye protection as a standard laboratory precaution.

Reagents Needed:

  • 20 mM NaOH
  • TE Buffer (10 mM Tris-HCl pH 8, 0.1 mM EDTA)

Bento Lab’s HotSHOT Extraction Buffer (25 mM NaOH) can be used instead of the 20 mM NaOH lysis buffer. The low EDTA TE neutralisation buffer can be approximated by a 5x dilution of the HotSHOT Neutralising Buffer (100 mM Tris-HCl, 0.5 mM EDTA, pH 8) in sterile distilled water, resulting in a 20 mM Tris-HCl pH 8, 0.1 mM EDTA solution.

Advantages:

  • Relatively rapid: 30 minutes incubation at room temperature plus drying time.
  • Cheap: Uses a dilute (20 mM) NaOH lysis buffer and washing in TE buffer.
  • Safe: 20 mM NaOH is unlikely to be harmful if handled with care and kept out of eyes.
  • Dried washed discs can be stored for direct PCR in the near future, allowing more convenient workflow organisation.

Disadvantages:

  • Not as rapid as other quick methods.
  • No extract stock is produced: each PCR uses a complete DBS disc.

Possible Issues:

  • No major issues identified for sheep, goats, or cows. The method has been occasionally used for human DBS. The protocol is still in use, e.g. in Ren et al. (2024) and  Xiang et al. (2023).

Protocol reference:

Zhou et al. (2006). A two-step procedure for extracting genomic DNA from dried blood spots on filter paper for polymerase chain reaction amplification. Analytical Biochemistry, 354(1), 159-161.

Examples references:

Ren et al. (2024). Expression and Variations in EPO Associated with Oxygen Metabolism in Tibetan Sheep. Animals, 14(4), 535.

Xiang et al.(2023). A simple alkali lysis method for Plasmodium falciparum DNA extraction from filter paper blood samples. Molecular and biochemical parasitology, 254, 111557.

Method 3: 75 °C Incubation in 0.2 M NaOH and Neutralisation (Rudbeck & Dissing, 1998)

This method was described by Rudbeck and Dissing (1998) for extraction of human genomic DNA for human tissues and forensic stains from whole blood, cheek cells, and semen. It uses a strong alkaline lysis followed by neutralisation, and has been used for a wide range of human and animal applications, including use for DNA extraction from feathers.

Protocol:

  1. Mix a blood spot containing around 5 µL of blood with 20 µL 0.2 M NaOH, and incubate at 75 °C for 5 min. Use a minimal amount of material to ensure it is fully soaked by the extraction buffer.
  2. Stop the extraction by adding 180 µL of 40 mM Tris-HCl pH 7.5. Use up to 2 µL of extract per 20 µL PCR.

Health and Safety Precautions:

0.2 M NaOH is very alkaline and caustic, and it requires careful handling and use even when used in very small volumes. Solid NaOH is extremely caustic and should be handled with great care. Be very careful to avoid spills and avoid contact with eyes and skin. Wear gloves and eye protection.

Reagents needed:

  • 0.2 M NaOH. This can be produced from solid NaOH by adding 8 g/L to sterile distilled/deionised water (or 0.24 g to 30 mL sterile distilled/deionised water for a convenient bottle format).
  • 40 mM Tris-HCl pH 7.5

Our HotSHOT DNA Extraction Buffer can be modified to 0.2 M NaOH by addition of 0.22 g of NaOH, and our HotSHOT Neutralisation Buffer can be diluted by 2.5x to create 75 mL of 40 mM Tris-HCl with 0.2 mM EDTA.

Advantages:

  • Rapid: 5 minute cell lysis.
  • Cheap: NaOH is inexpensive and 8 g can produce one L of 0.2M extraction buffer.
  • Simple and scalable: A two-step process that can be done entirely in PCR without centrifugation, allowing easy use of 8-strip PCR and 96-well PCR plate formats without compatible centrifuges.
  • A proven method: 0.2 M NaOH extractions have been widely used over the past few decades for many applications.

Disadvantages:

  • Produces single-stranded DNA which is less stable than double-stranded DNA.
  • The extraction will fragment DNA, which is not a problem for standard PCR but will be a problem for any methods requiring long unfragmented DNA.

Potential Issues:

None anticipated. Additional dilution of the crude extract may be needed for some samples.

Protocol Reference:

Rudbeck & Dissing (1998). Rapid, simple alkaline extraction of human genomic DNA from whole blood, buccal epithelial cells, semen and forensic stains for PCR. Biotechniques, 25(4), 588-592.

Method 4: 75 °C Incubation in 0.2 M NaOH, Neutralisation, and Centrifugation (Ramos-Díaz et al., 2015)

This method is a modification of Method 3 that was described and validated by Ramos-Díaz et al. (2015) for genotyping cancer patients from blood spots. It differs from Method 3 in the addition of 0.55 mM EDTA to the Neutralisation buffer to help preserve the extracted DNA from DNA-degrading enzymes, and includes a centrifugation step to remove any paper or cellular fragments.

Protocol:

  1. Add a 3-mm blood spot disc to 20 µL of 0.2 M NaOH, and incubate at 75 °C for 5 minutes.
  2. Stop the extraction by adding 180 µL of Tris-HCl 40 mM, pH 8, EDTA 0.55 mM, and mix vigorously. 
  3. Centrifuge at top speed for 5 minutes at room temperature to sediment any paper or cell remains.
  4. Transfer 170 µL of the supernatant into a different tube for immediate analysis, or store at -20 °C.

Health and Safety Precautions:

0.2 M NaOH is very alkaline and caustic, and requires careful handling and use even when used in very small volumes. Be very careful to avoid spills and avoid contact with eyes and skin. Wear gloves and eye protection.

Reagents needed:

  • 0.2 M NaOH. This can be produced from solid NaOH by adding 8 g/L to sterile distilled/deionised water (or 0.24 g to 30 mL sterile distilled/deionised water for a convenient bottle format).
  • 40 mM Tris-HCl pH 7.5

Our HotSHOT DNA Extraction Buffer can be modified to 0.2 M NaOH by addition of 0.22 g of NaOH, and our HotSHOT Neutralisation Buffer can be diluted by 2.5x to create 75 mL of 40 mM Tris-HCl with 0.2 mM EDTA.

Advantages:

  • Rapid: 10 minute extraction (5 mins lysis, 5 mins centrifugation).
  • Cheap: NaOH is inexpensive and 8 g can produce one L of 0.2M extraction buffer.
  • Simple: A two-step process that could be easily scaled to an 8-strip PCR tube format with a compatible centrifuge.
  • A proven method: 0.2 M NaOH extractions have been widely used over the past few decades for many applications, and the modification are very unlikely to decrease performance.
  • The DNA extract is probably more stable for long-term storage due to the removal of particles and addition of a small amount of EDTA.

Disadvantages:

  • An additional centrifugation step is needed which could slow down higher-throughput processing.
  • Produces single-stranded DNA which is less stable than double-stranded DNA.
  • The extraction will fragment DNA, which is not a problem for standard PCR but will be a problem for any methods requiring long unfragmented DNA.

Potential Issues:

None anticipated. Additional dilution of the crude extract may be needed for some samples.

Protocol Reference:

Ramos-Díaz et al. (2015). Validation of a fast and low-cost alkaline lysis method for gDNA extraction in a pharmacogenetic context. Cancer Chemotherapy and Pharmacology, 75, 1095-1098 (subscription access)

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