Buccal DNA Extraction & WGA Amplification Protocol

Introduction

This protocol provides a simple and convenient method to isolate, amplify, and purify genomic DNA from buccal swabs. Buccal swabs are a convenient method of acquiring a DNA sample. Once the DNA is isolated using the following extraction protocol, it can be amplified using the Whole Genome Amplification Protocol

It is recommended to use the GenElute Mammalian Genomic DNA Miniprep Kit (G1N10) for this process.

  1. Dry the collected swabs at room temperature for 15 minutes.
  2. Add 280 μl of Lysis Solution T and 20 μl of Proteinase K. Insert the swab and gently spin. Cap the tube and mix by vortexing.
  3. Incubate the sample at 55 °C for 20 minutes with occasional vortexing.
  4. Add 200 μl of Lysis Solution C and vortex thoroughly for 15 seconds.
  5. Incubate at 70 °C for 10 minutes.
  6. Add 500 μl of Column Preparation Solution to each GenElute Miniprep Binding Column (red o-ring) and centrifuge at 12,000 × g for 1 minute. Discard the flow-though liquid.
    Note: The Column Preparation Solution maximizes binding of DNA to the membrane resulting in more consistent yields
  7. Add 200 μl of 95-100% ethanol to the lysate from step 5.
  8. Mix thoroughly by vortexing and add the entire contents of the tube into the binding column.
  9. Centrifuge at ≥6500 × g for 1 minute.
  10. Discard the collection tube containing the flow-through and place the binding column in a new 2 ml collection tube.
  11. Add 500 μl of Wash Solution (be sure to dilute with ethanol prior to first use) and centrifuge at ≥6500 × g for 1 minute.
  12. Discard the collection tube and flow-through and place the binding column in a new 2 ml collection tube.
  13. Add another 500 μl of Wash Solution 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 binding column and centrifuge for 1 minute at ≥6500 × g.
  15. Store the eluted DNA at -20 °C or proceed to the amplification step.
    Note: If using WGA2 there is no need to supply DNA polymerase as the enzyme is provided with the kit

WGA Amplification

Protocol for GenomePlex Whole Genome Amplification performed with GenomePlex Whole Genome Amplification Kit (WGA1) and/or Complete Whole Genome Amplification Kit (WGA2).

Fragmentation

  1. Prepare DNA solution of 1 ng/ml from whole blood extraction protocol described above.
  2. Add 1 µl of 10X Fragmentation Buffer to 10 µl DNA (1 ng/µl) in a PCR tube.
  3. Place the tube in a thermal cycler at 95 °C for exactly 4 minutes. Note, the incubation is time sensitive and any deviation may alter results. 
  4. Immediately cool the sample on ice and centrifuge briefly.

Library Preparation

  1. Add 2 µl of 1x Library Preparation Buffer. 
  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 and centrifuge briefly.
  5. Add 1 µl Library Preparation Enzyme, mix thoroughly, and centrifuge briefly.
  6. Place sample in 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 up to three days.

Amplification

  1. Add the following reagents to the entire 15 µl reaction:
        7.5 µl 10x Amplification Master Mix
        47.5 µl Nuclease Free Water
        5.0 µl JumpStart Taq DNA Polymerase (12.5 units) for WGA1
        -or-
        5.0 µl WGA DNA Polymerase for WGA2
  2. Mix thoroughly, centrifuge briefly, and begin thermocycling
  3. After cycling is complete, maintain the reactions at 4 °C or store at –20 °C until ready for analysis or purification.

Reamplification

  1. Add 10 µl of 1 ng/ml WGA amplified DNA to a PCR tube or multiwell plate.
    Note: It is necessary to clean up the WGA reaction to decrease possible bias in the reamplification. We recommend using Sigma's GenElute™ PCR Clean-Up Kit (Product Number NA1020) or standard purification methods that isolate single and double stranded DNA.
  2. Create amplification mix. For each reamplification reaction, add the following to the WGA amplified DNA (step 1):
        47.5 µl of Nuclease-Free Water
        7.5 µl of 10X Amplification Master Mix
        5 µl of WGA DNA Polymerase
  3. 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 14 cycles as follows:
            Denature 94 °C for 15 seconds
            Anneal/Extend 65 °C for 5 minutes
  4. 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

Purification of Amplified Products performed with GenElute PCR Clean-Up Kit (NA1020)

  1. Insert a GenElute Miniprep Binding Column (with a blue O-ring) into a provided collection tube, if not already assembled. Add 0.5 ml of the Column Preparation Solution to each miniprep column and centrifuge at 12,000 x g for 30 seconds to 1 minute. Discard the eluate.
    Note
    : The Column Preparation Solution maximizes binding of the DNA to the membrane resulting in more consistent yields.
  2. Add 5 volumes of Binding Solution to 1 volume of the PCR reaction and mix. For example, add 500 µl of Binding Solution to 100 µl of the PCR reaction. Transfer the solution into the binding column. Centrifuge the column at maximum speed (12,000 to 16,000 x g) for 1 minute. Discard the eluate, but retain the collection tube.
  3. Replace the binding column into the collection tube. Apply 0.5 ml of diluted Wash Solution to the column and centrifuge at maximum speed for 1 minute. Discard the eluate, but retain the collection tube.
    Note
    : Be sure to add ethanol to the Wash Solution Concentrate prior to first time use. See Preparation Instructions.
  4. Replace the column into the collection tube. Centrifuge the column at maximum speed for 2 minutes, without any additional wash solution, to remove excess ethanol. Discard any residual eluate as well as the collection tube.
  5. Transfer the column to a fresh 2 ml collection tube. Apply 50 µl of Elution Solution or water to the center of each column. Incubate at room temperature for 1 minute.
    Note
    : When eluting with water, make sure that the pH of the water is between 5.5 and 8.5. Elution may also be performed using the Elution Solution diluted 10-fold with water.
  6. To elute the DNA, centrifuge the column at maximum speed for 1 minute. The PCR amplification product is now present in the eluate and is ready for immediate use or storage at –20 °C.

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/

Application Data

Whole Genome Amplification Performed on a Buccal Swab Sample

Lane 1 - 1 kb Ladder
Lane 2 - Blood
Lane 3 - Plant
Lane 4 - Buccal Swab
Lane 5 - Soil
Lane 6 - Positive Control
Lane 7 - 1 kb Ladder

Materials

     

Materials to be Supplied by User

  • Buccal Swab
  • 1.5 ml microcentrifuge tubes
  • Microcentrifuge (with rotor for 2 ml tubes)
  • 55 °C water bath or heat block

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