Experimental purification of PCR products with dUTP

Summary

The incorporation of dUTP into the replicon during PCR amplification is one of the most efficient and widely used methods for purifying PCR products (Longoetal.1990;HartleyandRashtchian1995). The detailed procedure for this method is described below. This experiment was derived from PCR Laboratory Guide (2nd ed.) by Seed Kang and Qu Lijia.

Operation method

Experimental purification of PCR products with dUTP

Materials and Instruments

dNTP solution MgCl2 solution PCR buffer Primers Uracil-DNA-glycosylase Taq DNA polymerase
PCR Instrument Centrifuge Tubes

Move

I. Materials

1. Reagents

(1) dNTP solution (containing dATP, dCTP, dGTP, and dUTP, each at a concentration of 10 mmol/L) In most cases, direct replacement of dUTP is sufficient (RySandPersingl993;Koxetal.1994). If necessary, the final dUTP concentration can be increased to 0.6 to 1 mmol/L to compensate for inhibition of the polymerase (Wangetal.1992;Hohlfeldetal.1994).

(2) MgCl₂ solution, 50 mmol/L

A final ^Mg2+ concentration of 1.5 mmol/L is sufficient in most cases, however, it should be optimized according to nucleotide (e.g., dUTP) concentration and primer specificity. Increasing the nucleotide concentration should be accompanied by increasing the ^Mg2+ concentration.

(3) PCR buffer, 10X (100 mmol/L Tris-HCl, pH 8.4, 500 mmol/L KCl)

2. Primers

Forward primer (l0umol/L)

Reverse primer (l0umol/L)

3. Enzyme

(1) uracil-DNA-glycosylase, 1U/ul (Invitrogen)

1 UDG removes 5 ng of contaminant, which can be reduced to 0.01 activity U (KoxetaL1994)B depending on the level of purification required (2) Taq DNA polymerase 5U/ul

Hot Start Platinum7' Called DNA Polymerase Invitrogen10966-018) Allows preparation of PCR reaction solutions at room temperature.

4. Equipment

A PCR instrument that can be programmed (note the modifications in (3) and (4) in "II. Methods") and thin-walled centrifuge tubes for PCR amplification.

Methods

(1) Add the following reagents to a sterilized centrifuge tube on ice.

10 x PCR buffer 5ul

dNTP solution 1 ul
Note: dNTP solution containing dATP, dCTP, dGTP and dUTP at 10 mmol/L each.

Forward primer, reverse primer 1 ul (l0umol/L each) uracil-DNA-glycosidase 1U/ul 1U

TagDNA polymerase (5U/ul) 0.5ul

MgCl₂ (50 mmol/L) 1.5ul

_H20 40ul

(2) Incubate at 37°C for l0min.

This UDG step removes fecal pyrimidine residues from the contaminant. It has been reported that this purification process can also be done at room temperature (dcWitetal.1993; Wangetal.1992).

(3) Heat at 94°C for l0min to inactivate UDG and hydrolyze the contaminating DNA (Hohlfeldetal.1994; Koxetal.1994).

(4) Add 2 cycles to the standard PCR.

The addition of 2 cycles can compensate for the effect of dUTP on amplification efficiency (Longoetal.1990).

(5) Keep the temperature at 72°C until the end of the PCR, until storage.

It has been reported that the Escherichia coli UDG recovers a small portion of its catalytic activity if the temperature is lowered after the end of PCR.

(6) Store the reaction mixture containing uracil DNA as well as UDG activity at -20°C.

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