Chapter 10 : DNA Extraction From Sperm Samples

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DNA extraction from sperm samples is a crucial technique in various fields such as forensic science, paternity testing, and fertility research. The silica column method provides a reliable and efficient way to isolate high-quality DNA from sperm cells. This blog post will outline the general procedure for DNA extraction from sperm samples using a silica column.

Materials Required

  • Sperm sample
  • Lysis buffer
  • Proteinase K
  • Binding buffer
  • Wash buffer
  • Elution buffer
  • Silica columns
  • Collection tubes
  • Microcentrifuge

Procedure

  • Sample Preparation and Lysis:
    • If the sperm sample is in a semen or other fluid, it may need to be washed and centrifuged to concentrate the sperm cells.
    • Add lysis buffer to the sperm sample. This buffer contains detergents and enzymes, such as Proteinase K, that break down cell membranes and proteins, releasing DNA into the solution.
    • Incubate the sample at an appropriate temperature to ensure complete lysis
  • DNA Binding to Silica Column:
    • Add binding buffer to the lysate. This buffer creates conditions that promote DNA binding to the silica membrane within the column.
    • Transfer the lysate to a silica column.
    • Centrifuge the column to bind DNA to the membrane and remove impurities.
  • Washing:
    • Wash the silica column with wash buffer to remove any remaining contaminants.
    • Centrifuge the column to remove the wash buffer.
  • DNA Elution:
    • Add elution buffer (low salt buffer or water) to the silica column.
    • Centrifuge the column to elute the purified DNA into a collection tube.

Tips and Considerations

  • Handle sperm samples with care and follow appropriate safety guidelines.
  • Ensure all reagents and equipment are RNase-free to prevent RNA contamination.
  • Optimize lysis and binding conditions for efficient DNA extraction from sperm cells.
  • Consider using specialized sperm lysis buffers if available.

Applications of Extracted Sperm DNA

The DNA extracted from sperm using the silica column method can be used for various applications, including:

  • Paternity testing
  • Forensic analysis
  • Fertility research
  • Genetic testing

Conclusion

Silica column-based DNA extraction from sperm samples is a valuable tool in various fields. By following the outlined steps and considering the provided tips, you can successfully isolate high-quality DNA from sperm for your downstream applications.

Lysis Buffer :

DNA lysis buffer is a solution that breaks open blood cells to extract DNA. It’s used in molecular biology experiments to analyze DNA.

Binding Buffer :

A binding buffer is a reagent used to bind DNA to silica during DNA extraction. It helps to purify DNA from various samples, including blood, cells, and tissues. It contains Guanidine Hydrochloride and Nuclease free water

DNA Wash Buffer :

(80% ethanol, 20 mM NaCl, 2 mM Tris-HCl, pH 7.5)

Elution Buffer :

(0.5 mM EDTA, 10 mM Tris-HCl, pH 9.0 )

 DNA Extraction Protocol

  • Take 100-250μl sperm sample and transfer it to 1.5 ml microfuge tube.
  • Add 0.2 ml elution buffer to the microfuge tube, Vortex for 10 seconds at full speed.
  • Centrifuge at 5000 rpm for 5 minute.
  • Carefully remove the supernatant, leaving the pellet.
  • Re-suspend the pellet in 200 μl Lysis Buffer and mix it well by vortexing for 15 seconds.
  • Add 20 μl of Proteinase K and incubate for 15-20 minutes at 60°C. Mix lysate after interval of 5 minutes by inverting the tube.
  • Note: Ensure that 20 μl of Proteinase K has been added to Lysis buffer as instructed. 
  • After incubation add 1 volume of binding buffer and 0.5 volume of 96-100 % ethanol. (For example Add 200 μl of Binding Buffer  + 150 μl 96-100 % ethanol for 200 μl of Lysis Buffer )and mix well with inverting tubes 20 times .
  • Transfer entire lysate to the DNA column, and centrifuge at 10000 rpm for 1 min. Discard the flow through liquid.
  • Repeat above step, until the entire sample has been processed and retain column for further processing.
  • Place the column into same collection tube. Add 500μl of Wash buffer. Centrifuge at 10,000 rpm for 1 min. discard the flow through.
  • Repeat the above step for removal of impurities.
  • Place empty DNA spin column, with the lid open into the same collection tube and centrifuge at 12,000 rpm for 2 min.
  • Place the column into a new sterile 1.5 ml eppendorf tube, add 30 μl preheated Elution buffer. Incubate at room temperature for 5 min. (perform this step twice 30 μl +30μl=60μl) 
  • Centrifuge 10,000 rpm for 1 min to elute pure sperm gDNA. The first elution normally yields 60-70 % of DNA bound. A second elution with another 30 μl buffer will yield another 20 % of the DNA. 
  • Note: Elution volume may vary as per downstream process 
  • Discard the Column, and save elute. Do not reuse binding columns or collection tubes. 

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