Whole Genome Amplification

Whole Blood


Whole blood is a common source of material used to perform genetic analysis. Many times genomic DNA isolated from whole blood samples is of low yield. This can hinder the researcher’s ability to perform downstream analysis. The following protocol is a simple method to isolate DNA from fresh or aged whole blood products. Once the DNA is isolated, it can be amplified using the GenomePlex® Whole Genome Amplification protocol.

Required Products

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

 

Materials to be Supplied by the User

  • Whole card
  • 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 a Whole Blood
The GenElute Blood Genomic DNA Kit (Product No. NA2000) is recommended for this procedure.

  1. Place 20 µl of Proteinase K into a 1.5 ml microcentrifuge tube and add 200 µl of whole blood to the tube.
  2. Add 200 µl of Lysis Solution C and vortex thoroughly for 15 seconds.
  3. Incubate at 55 °C for 10 minutes.
  4. 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.
  5.  
  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.  
  13. 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.
  14. Pipette 200 µl of Elution Solution onto the column and centrifuge for 1 minute at ≥6500 × g to elute the DNA.
  15. Store the eluted DNA at –20 °C or proceed with the amplification step.

 

 

Application Data

Human Whole Blood Amplified DNA – Comparison Data

Whole Blood

 

 

Legend: Products were amplified using the GenomePlex Whole Genome Amplification Kit (Product No. WGA1) from Sigma, Supplier A’s kit and Supplier Q’s kit. Products were resolved on a 1.5% agarose gel. 5 µl of amplified product was added to each well. The products amplified using GenomePlex technology were of a smaller molecular weight as shown on the gel when compared to Supplier A and Q. This is due to the random fragmentation of genomic DNA prior to amplification. Sigma’s amplified products are specific and there is no amplicon visible in the negative control (lane 2) indicating that only the desired genomic DNA is amplified. Both Suppliers A and Q yield a nonspecific signal in the negative control which is equal in size and intensity to the signal for the suppliers’ positive control.

Lane 1—1 kb Marker
Lane 2—Sigma Positive Control
Lane 3—Sigma Negative Control
Lane 4—Sigma Blood Card
Lane 5—Sigma Blood Card
Lane 6—Supplier A Positive Control
Lane 7—Supplier A Negative Control

Lane 8—Supplier A Blood Card
Lane 9—Supplier A Blood Card
Lane 10—Supplier Q Positive Control
Lane 11—Supplier Q Negative Control
Lane 12—Supplier Q Blood Card
Lane 13—Supplier Q Blood Card
Lane 14—1 kb Marker

 

Performance of DNA Amplified with GenomePlex WGA Identical to Non-Amplified DNA

Genomic DNA Charts

 

Legend: Products were amplified using the GenomePlex Whole Genome Amplification Kit (Product No. NA2000). 10 ng of genomic DNA was amplified using the GenomePlex WGA Kit (Product No . WGA1) followed by purification using the GenElute PCR Clean-up Kit (Product No. NA1020). SNP genotyping analysis was performed on non-amplified DNA and GenomePlex amplified DNA. GenomePlex WGA DNA genotyping provided the same accuracy and quality of scores to non-amplified DNA, indicating that the amplification process did not alter the original genomic sequence.

 

Back to Top