WGA Prep

Whole Genome Amplification Notes
Our group uses whole genome amplification (WGA, or MDA) to amplify rare DNAs, as well as to prepare large quantities of biotinylated 'concaterpiller' probes (see the page on Illumina GA Hyb-Seq for details). The reagents we use are shown below.

Note: We use the New England enzymes in our own laboratory, but this is not an endorsement of their products; we expect that similar results could be obtained with any standard molecular biology grade enzyme source.

1. MDA Reagents

• Φ29 polymerase and buffer; NEB M0269OL (250 units, 10U/μL)

• Trehalose (D-(+)-trehalose dehydrate, Sigma T5251). Pan et al. (2008) show that the addition of trehalose to MDA reactions minimizes the formation of template-independent products.

• Yeast inorganic pyrophosphatase, NEB M2403S (10 U, 0.1U/μL). Dean et al. (2001) include this enzyme; may be necessary to prevent end-product inhibition.

• Random hexamers, nuclease protected. We order from IDT (1 μmol). Dilute to a stock concentration of 1 nmol/μL (= 1000 μM). Note: To make nuclease resistant oligonucleotides, 3’ phosphodiester bonds are replaced with phosphorothioate bonds. From IDT, this is specified using the notation 5’-NNNN*N*N-3OH’ (* = phosphorothioate). We modify two bonds because the product at each coupling is a 1:1 racemic mix. Minimum order size for this oligo is 1 μmol.

2. Template Denaturation

• Read Dean et al. (2001) very carefully before you denature. Kits simplify this by making the decision for you, but their decision may not be the best choice. Choices include:

    o Base denaturation

	Prepare Solutions D and N as indicated (see below)

	Mix 1 μL DNA mixture (between 0.1 and 10 ng) with 1 μL Solution D.

	Mix well; incubate at RT for 3 minutes.

	Neutralize by addition of 2 μL Solution N.

	Add 1 μL random hexamer stock, then chill on ice.

    o Heat denaturation (not recommended by Dean):

	Mix 4 μL of DNA mixture (0.1 and 10 ng) with 1 μL of random hexamer stock.

	Heat samples to 80°C for 2 minutes, then cool at rate of 4 degrees/minute to 20°C.

	Chill on ice.

    o No denaturation (this works for many templates):

	Mix 4 μL of DNA mixture (0.1 and 10 ng) with 1 μL of random hexamer stock.

	Chill on ice.

3. Reaction details

• Incubation temperature is 30°C; incubation time is 18 hours

• A typical 20 μL reaction cocktail using NEB reagents contains:

o	2 μL 10X NEB reaction buffer

o	2 μL 0.5 M KCl

o	2 μL of 10 mM dNTPs

o	5 μL of 0.8 M trehalose stock (diluted in d.i. water)

o	3.5 μL water

o	5 μL DNA/random hexamer mixture (denatured or not)

o	0.5 μL Φ29 polymerase (5 units)

4. Notes

• Be sure to limit the input DNA to 10 ng. We’ve seen some evidence of reaction inhibition at high DNA concentrations.

• Solutions D and N were prepared as follows:

 1X D = 0.4 M KOH, 10 mM EDTA. Make this as an 8X stock (3.2 M KOH, 80 mM EDTA). It's only stable for 6 months at -20°C storage. Dilute to 1X each time before use.

 1X N = 0.4 M Trizma-HCl, pH unadjusted. Make this as a 10X stock (4 M Trizma-HCL). In contrast to Solution D, this reagent is stable at -20°C. Dilute to 1X, and then save in the freezer between uses.

• 10 mM dNTP: make or purchase from your favorite supplier.

• NEB 10X Φ29 buffer: This contains DTT so protect it from repeated freezing and thawing. When thawed the first time, we aliquot this buffer at 50 μL per tube (into many tubes).

• Yes, reactions can be scaled back further if you like. We’ve had success with 10 μL and 5 μL reaction volumes.

The cost of these reagents from NEB and IDT •	1 minimum order (50 samples) = $1.01 per 20 μL reaction

Sources For This Method

 * 1) Paper1 pmid=11959976
 * 2) Paper2 pmid=18832167
 * 3) Paper3 pmid=16928277
 * 4) Paper4 pmid=18832167

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