Troubleshooting for DNA extraction from blood

Troubleshooting for DNA extraction from blood

Troubleshooting DNA extraction from blood can be challenging, but here are some common issues and potential solutions:

Common Issues and Solutions:

1. Low Yield:

   • Cause: Incomplete cell lysis, old blood samples, or improper storage.
   • Solution: Ensure thorough lysis by increasing incubation time with lysis buffer, using a more aggressive lysing matrix, or adding Proteinase K and RNase A to the frozen samples. Use fresh blood samples and avoid thawing them before extraction1.

2. DNA Degradation:

   • Cause: DNase activity in thawed blood samples.
   • Solution: Keep blood samples frozen until the extraction process begins and add lysis buffer and enzymes directly to the frozen samples.

3. Contamination:

   • Cause: Hemoglobin precipitates or other contaminants.
   • Solution: Reduce Proteinase K lysis time to prevent hemoglobin precipitate formation. Centrifuge lysate to remove protein precipitates before applying the sample to a spin filter1.

4. Difficulty Resuspending DNA Pellet:

   • Cause: Insufficient resuspension techniques.
   • Solution: Use cold PBS for resuspension and gently pipette up and down to achieve a uniformly turbid suspension.

5. Clogged Filters:

   • Cause: Fibrous tissue or protein precipitates.
   • Solution: Centrifuge lysate at maximum speed to remove fibers and protein precipitates. For fibrous tissues, cut them into smaller pieces or grind with liquid nitrogen2.

General Tips:

• Proper Storage: Keep blood samples frozen until the extraction process begins.
• Fresh Samples: Use fresh blood samples, ideally within a week.
• Optimize Lysis: Adjust lysis buffer and enzyme concentrations as needed.

By addressing these common issues, you can improve the yield and quality of DNA extracted from blood samples.

Let’s dive into some specific problems you might encounter during DNA extraction from blood and how to troubleshoot them:

Specific Problems and Solutions:

1. Incomplete Lysis

• Problem: Blood cells are not fully lysed, resulting in low DNA yield.
• Solution:
  • Increase the incubation time with the lysis buffer.
  • Use a more aggressive lysing buffer or additional enzymes like Proteinase K.
  • Ensure proper mixing and possibly increase the temperature slightly during the lysis step.

2. Low DNA Yield

• Problem: Insufficient amount of DNA is extracted.
• Solution:
  • Ensure the blood sample is fresh and stored properly (preferably frozen if not used immediately).
  • Use an adequate volume of lysis buffer relative to the sample size.
  • Verify the efficiency of the cell lysis step; consider a different lysis buffer if necessary.
  • Check the DNA precipitation step and ensure all reagents (like ethanol or isopropanol) are fresh and at the correct concentrations.

3. DNA Degradation

• Problem: Extracted DNA is degraded, leading to poor quality.
• Solution:
  • Use fresh or properly stored blood samples to minimize DNase activity.
  • Add DNase inhibitors if necessary.
  • Handle samples gently to prevent mechanical shearing.
  • Work quickly and keep samples on ice when possible.

4. Contamination

• Problem: Presence of contaminants (e.g., proteins, hemoglobin) that can inhibit downstream applications.
• Solution:
  • Use phenol-chloroform extraction to remove proteins.
  • Include an additional washing step with 70% ethanol to remove contaminants.
  • Ensure proper centrifugation to pellet debris and contaminants.

6.       Clogged Filters or Columns

• Problem: Filters or columns used in purification steps get clogged, reducing yield.
• Solution:
  • Centrifuge lysates at high speed to remove cell debris before loading onto the column.
  • Pre-filter the sample using a low-speed spin to remove particulates.
  • Consider using larger pore-size filters if clogging persists.

6. Inhibitors in PCR

• Problem: Inhibitors from the blood (like heme) affect the PCR reaction.
• Solution:
  • Purify the DNA using additional steps such as silica-based column purification or ethanol precipitation.
  • Use PCR additives like BSA or betaine to mitigate inhibition.
  • Increase the number of washing steps to remove potential inhibitors.

General Tips:

• Sample Quality: Always start with high-quality, fresh blood samples.
• Consistent Protocols: Stick to optimized and validated protocols for your specific application.
• Regular Monitoring: Check the DNA concentration and purity using spectrophotometry (260/280 ratio) after extraction.