Whole Genome Amplification

Serum Plasma

Whole genome amplification (WGA) of plasma and serum DNA presents a unique challenge due to the small amount of nucleic acid in such samples. One must use a robust DNA purification scheme capable of removing inhibitory substances, while at the same time, concentrating the DNA within the sample. The use of GenElute™ Blood Genomic DNA Kit in combination with one of two modified WGA protocols has been shown to work well with DNA isolated from serum or plasma. Both procedures typically produce approximately 5 µg of WGA product. Old serum and plasma samples may contain degraded DNA, and therefore may not be suitable for downstream applications such as WGA. The WGA procedures described below are modifications of the WGA2 and WGA4 protocols. The procedures work equally well, each typically producing approximately 5 µg of WGA product.

Required Products

  • GenElute™ Blood Genomic DNA Kit (Product No. NA2000)

 

Materials to be Supplied by the User

  • Serum or Plasma
  • 1.5 ml microcentrifuge tubes
  • Ethanol (Product No. E7023)
  • Microcentrifuge (with rotor for 2 ml tubes)
  • Water, molecular biology reagent (Product No. W4502)
  • 55 °C water bath or heat block

 

Extraction of DNA from Serum or Plasma
The GenElute Blood Genomic DNA Kit (Product No. NA2000) is recommended for this process. It is recommended to start with 0.1 ml to 1 ml serum or plasma. The protocol below is written for 0.1 ml to 0.2 ml serum or plasma. Scale GenElute reagents proportionally if using more than 0.2 ml serum or plasma.

  1. Cut a disc from a dried blood card (200 µl spotted) into several 2 mm by 2 mm pieces and place the pieces into a 1.5 ml microcentrifuge tube.
  2. Place 20 µl of Proteinase K into a 1.5 ml microcentrifuge tube and add 200 µl of plasma or serum to the tube.
  3. Add 200 µl of Lysis Solution C and vortex thoroughly for 15 seconds.
  4. Incubate at 55 °C for 10 minutes.
  5. Add 500 µl of Column Preparation Solution to the GenElute Miniprep Binding Column (red o-ring) and centrifuge at 12,000 × g for 1 minute.
    Note: The Column Preparation Solution maximizes binding of DNA to the membrane resulting in more consistent yields.
  6. Discard the flow-through liquid.
  7. Add 200 µl of 95–100% ethanol to the lysate from step 3 and mix thoroughly by vortexing 5–10 seconds.
  8. Transfer the entire contents of the tube into the treated column from step 4. Centrifuge at 6500 × g for 1 minute.
  9. Discard the collection tube and flow-through. Place the column into a new 2 ml collection tube.
  10. Add 500 µl of Prewash Solution (be sure to dilute with ethanol prior to first use) and centrifuge at 6500 × g for 1 minute.
  11. Discard the collection tube containing the flow-through and place the column into a new 2 ml collection tube.
  12. Add 500 µl of Wash Solution (be sure to dilute with ethanol prior to first use) to the binding column and centrifuge at maximum speed (12,000–16,000 × g) for 3 minutes to dry the binding column.
  13. Pipette 50 µl of Elution Solution onto the column and centrifuge for 1 minute at 6500 × g to elute the DNA.
  14. Store the eluted DNA at –20 °C or proceed to the amplification step with WGA2 or WGA4.

 

Amplification with WGA2

  1. Combine 10 µl of serum or plasma DNA, purified as outlined above, and 1 µl of 10× Fragmentation Buffer to a PCR tube or multiwell strip/plate.
    Note: Use 10 µl of DNA regardless of concentration, since spectrophotometric quantitation of very dilute DNA is not accurate. 10 µl is the maximum volume that can be used in WGA.
  2. Continue with step 3 of the WGA2 protocol, however, perform 25 amplification cycles in step 13, as opposed to 14 cycles.

Amplification with WGA4

  1. Combine 10 µl of serum or plasma DNA, purified as outlined above, and 1 µl of 10× Single Cell Lysis & Fragmentation Buffer to a PCR tube or multiwell strip/plate. Mix thoroughly.
    Note: Use 10 µl of DNA regardless of concentration, since spectrophotometric quantitation of very dilute DNA is not accurate. 10 µl is the maximum volume that can be used in WGA.
  2. Place the tube/plate in a thermal block or cycler at 95 °C for EXACTLY 4 minutes. Immediately cool on ice. Spin down sample prior to proceeding to Library Preparation.
    Note: This incubation is very time sensitive. Any deviation may alter results.
  3. Continue with the Library Preparation section (step 6) of the WGA4 technical bulletin (152 Kb PDF).

     

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