Whole Genome Amplification from Serum or Plasma Protocol

Introduction

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.

The GenElute Blood Genomic DNA Kit (NA2010) 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. Place 20 μL of Proteinase K into a 1.5 mL microcentrifuge tube and add 200 μL of plasma or serum 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. Discard the flow-through liquid.
6. Add 200 μL of 95–100% ethanol to the lysate from step 3 and mix thoroughly by vortexing 5–10 seconds.
7. Transfer the entire contents of the tube into the treated column from step 4. Centrifuge at 6500 × g for 1 minute.
8. Discard the collection tube and flow-through. Place the column into a new 2 mL collection tube.
9. Add 500 μL of Prewash Solution (be sure to dilute with ethanol prior to first use) and centrifuge at 6500 × g for 1 minute.
10. Discard the collection tube containing the flow-through and place the column into a new 2 mL collection tube.
11. 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.
12. Pipette 50 μL of Elution Solution onto the column and centrifuge for 1 minute at 6500 × g to elute the DNA.
13. 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 multiwall 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.

WGA2 Protocol

1. Place the tube/plate in a thermal block or cycler at 95 °C for EXACTLY 4 minutes. Note, the incubation is very time sensitive. Any deviation may alter results.
2. Immediately cool the sample on ice, then centrifuge briefly to consolidate the contents

Library Preparation

1. Add 2 µL of 1x Library Preparation Buffer to each sample.
2. Add 1 µL of Library Stabilization Solution.
3. Vortex thoroughly, consolidate by centrifugation, and place in thermal cycler at 95 °C for 2 minutes.
4. Cool the sample on ice, consolidate the sample by centrifugation, and return to ice.
5. Add 1 µL of Library Preparation Enzyme, vortex thoroughly, and centrifuge briefly.
6. Place sample in a thermal cycler and incubate as follows:
   • 16 °C for 20 minutes
   • 24 °C for 20 minutes
   • 37 °C for 20 minutes
   • 75 °C for 5 minutes
   • 4 °C hold
   • Remove samples from thermal cycler and centrifuge briefly. Samples may be amplified immediately or stored at –20 °C for three days.

Amplification

1. A master mix may be prepared by adding the following reagents to the 15 µL reaction from step 11:
   • 7.5 µL of 10X Amplification Master Mix
   • 47.5 µL of Water, Molecular Biology Reagent
   • 5 µL of WGA DNA Polymerase
2. Vortex thoroughly, centrifuge briefly, and begin thermocycling. The following profile has been optimized for a PE 9700 or equivalent thermocycler:
   • Initial Denaturation 95 °C for 3 minutes
   • Perform 25 cycles as follows:
   • Denature 94 °C for 15 seconds
   • Anneal/Extend 65 °C for 5 minutes

After cycling is complete, maintain the reactions at 4 °C or store at –20 °C until ready for analysis or purification. The stability of WGA DNA is equivalent to genomic DNA stored under the same conditions.

Purification of the final product is recommended before use in subsequent applications. WGA amplified DNA may be purified with our PCR Cleanup Kit (Catalog Number NA1020) or standard purification methods that isolate single and double stranded DNA. Once purified, the DNA can be quantified by measuring absorbance, assuming that one A260 unit is equivalent to 50 ng/µL DNA. Measurement techniques such as PicoGreen^® will often underestimate the actual WGA DNA yield, since single stranded DNA might be generated during amplification.

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 multiwall 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.

Library Preparation (WGA4)

1. Add 2 µL of 1x Single Cell Library Preparation Buffer to each sample.
2. Add 1 µL of Library Stabilization Solution.
3. Mix thoroughly and place in thermal cycler at 95 °C for 2 minutes.
4. Cool the sample on ice, consolidate the sample by centrifugation, and return to ice.
5. Add 1 µL of Library Preparation Enzyme, mix thoroughly, and centrifuge briefly.
6. Place sample in a thermal cycler and incubate as follows:
   • 16 °C for 20 minutes
   • 24 °C for 20 minutes
   • 37 °C for 20 minutes
   • 75 °C for 5 minutes
   • 4 °C hold
7. Remove samples from thermal cycler and centrifuge briefly. Samples may be amplified immediately or stored at –20 °C for three days.

Amplification

1. Add the following reagents to the entire 14 µL reaction:
   • 7.5 µL of 10x Amplification Master Mix
   • 48.5 µL of Water, Molecular Biology Reagent
   • 5 µL of WGA DNA Polymerase
2. Mix thoroughly, centrifuge briefly, and begin thermocycling. The following profile has been optimized for a PE 9700 or equivalent thermocycler:
   • Initial Denaturation  95 °C for 3 minutes
   • Perform 25 cycles as follows:
   • Denature 94 °C for 30 seconds
   • Anneal/Extend 65 °C for 5 minutes
   • Hold 4 °C

After cycling is complete, maintain the reactions at 4 °C or store at –20 °C until ready for analysis or purification. The stability of WGA DNA is equivalent to genomic DNA stored under the same conditions.

Purification of the final product is recommended before use in subsequent applications. WGA amplified DNA may be purified with GenElute™ PCR Cleanup Kit (Catalog Number NA1020) or standard purification methods that isolate single and double stranded DNA. Once purified, the DNA can be quantified by measuring absorbance, assuming that one A260 unit is equivalent to 50 ng/µL DNA. Measurement techniques such as PicoGreen^® dye will often underestimate the actual WGA DNA yield, since single stranded DNA might be generated during amplification.

Quantification of Amplified Products

The amount of DNA amplified using Whole Genome Amplification can be detected with or without purification. For the highest quality samples of DNA, it is strongly recommended to purify the samples after amplification. The amplified products can be measured with the PicoGreen^® dsDNA Quantitation Assay from Molecular Probes Inc. (#P-7589). Another method of detecting the amplified products is spectrophotometric absorption (OD260) on a NanoDrop^® spectrophotometer. This instrument can measure absorbance on 1 μL of sample over a large dynamic range, from 2–3700 ng/

Materials to be Supplied by User

• Serum or Plasma
  
• 1.5 mL microcentrifuge tubes
• Microcentrifuge (with rotor for 2 mL tubes)
• 55 °C water bath or heat block