Saliva DNA Extraction & WGA Amplification Protocol

Introduction

Whole Genome Amplification can be performed on DNA extracted in many ways. We offer many products for DNA extraction, including the GenElute™ Blood Genomic DNA Kit (NA2010), GenElute Mammalian Genomic DNA Miniprep Kit (G1N10), and the GenElute Plant Genomic DNA M iniprep (G2N10). Genome- Plex Whole Genome Amplification has also been used to amplify DNA from soil and saliva samples. DNA from saliva can be extracted using DNA Genotek’s Oragene™ DNA Self-Collection Kit. DNA from soil can be extracted using the UltraClean™ Soil Kit by Mo Bio Laboratories, Inc. (#12800-50). The protocols are included below.

This protocol provides a simple and convenient method to isolate, amplify, and purify genomic DNA from saliva. It is recommended to use the Oragene™ DNA Self-Collection Kit from DNA Genotek, Inc. for this procedure.

Purification

Purification from 500 μL aliquot

1. Incubate the Oragene/saliva sample in the Oragene vial at 50 °C in a water bath or in an air incubator for a minimum of 1 hour. The sample may be incubated overnight if this is more convenient.
2. Transfer 500 μL of the Oragene/saliva sample to a 1.5 mL microcentrifuge tube. The remainder of the Oragene/saliva sample can be stored at room temperature until ready for further use.
3. Add 20 μL (or 1/25^th of the total volume) of Oragene Purifier (supplied with kit) and mix gently by inversion. The sample will become turbid as impurities are precipitated.
4. Incubate on ice for 10 minutes.
5. Centrifuge for 3 minutes at 13,000 rpm at room temperature. Carefully pipette the clear supernatant into a fresh microcentrifuge tube without disturbing the pellet. Discard the pellet.
6. Add 500 μL (or an equal volume) of room temperature 95% ethanol to the supernatant and mix gently by inverting at least 5 times. A clot of DNA may be visible.
7. Let the solution stand for 10 minutes at room temperature so that the DNA is fully precipitated. Do not incubate at –20 °C because impurities will co-precipitate with the DNA.
8. Centrifuge for 1 minute at 13,000 rpm at room temperature. Discard the supernatant without disturbing the DNA pellet (may or may not be visible).
9. If necessary, centrifuge again for 10 seconds to remove excess ethanol.
10. Once all of the ethanol has been removed, dissolve the DNA pellet in 100 μL of TE buffer or other standard buffer. The expected concentration of the rehydrated DNA is 10 to 100 ng/μL.
11. To fully dissolve the DNA, vigorous vortexing followed by incubation for a minimum of 1 hour at room temperature or preferably overnight, is recommended Alternatively, incubation for 10 minutes at 50 °C is also effective.
12. Store the 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/μL 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:
   Initial Denaturation: 95 °C for 3 minutes
   Perform 14 cycles as follows:
   Denature: 95 °C for 15 seconds
   Anneal/Extend: 65 °C for 5 minutes
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 our GenElute™ PCR Clean-Up Kit (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/

Materials to be Supplied by User

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