Phosphorylation of the solid phase primer

  1. Introduction
  2. Phosphorylation of the 5'-end of the primer
    1. Determination of purity and concentration: OD260 and OD280
  3. Reagents
    1. DNA
    2. 10 kinase buffer
    3. 1 M Tris-HCl7.6
    4. 0.3 M MgCl2
    5. 0.5 M DTT
    6. 0.01 M Sodium acetate - pH 5.2
    7. ATP
  4. Conclusion

1. Introduction
The solid phase primer must be phosphorylated in the 5'-end in order to be covalently bound to the surface of the NucleoLink Strips ( EDC coupling reaction). Furthermore, it is very important that the solid phase primer is prepared with a linker in the 5'-end ( A linker on the 5' - end of the solid phase primer). Linkers of 10-20 thymidine residues have been successfully used at the Nunc A/S Research Laboratory. The solid phase primer can be 5'-end phosphorylated either during synthesis of the primers or in a subsequent kinase reaction where a phosphate group is added ( Phosphorylation of the 5'-end of the primer).

A linker is necessary to achieve a good solid phase DNA amplification ( DIAPOPS results as a function of a linker in the 5'-end of the solid phase primer), and it is therefore NOT feasible to directly phosphorylate a liquid phase primers and use it as a solid phase primer. If the liquid phase primer is very long (over 25 bases), results have shown that they can perform reasonably well as solid phase primers in the DIAPOPS analysis in some systems. This demands that they are directly phosphorylated. Since this has only been seen in a few systems it is not recommended. The kinase reaction should therefore only be used if the primer previously have been modified with this linker. It is recommended that the the solid phase primer is ordered with both the linker and the phosphate group in the 5'-end.

2. Phosphorylation of the 5'-end of the primer
A long primer or a primer already containing a linker ( DIAPOPS results as a function of a linker in the 5'-end of the solid phase primer), but not having the phosphate group necessary for covalent binding to the surface of NucleoLink Strips ( EDC coupling reaction), can be phosphorylated in the 5'-end using the following procedure.

The water used has been purified on a Milli-Q PLUS 185 device, which is also fitted with a sterile filter. In the procedure the water is called "Milli-Q water". The reaction volume is large (5 ml), because the amount of phosphorylated oligo needed is large (100 ng for one well).

A mix of the following components is prepared:

200 µl 50 µM oligo DNA to be 5'-end phosphorylated (approx. 80 µg)
500 l 10 kinase buffer
100 l 1 nmol/l ATP dilution
10 l T4 Polynucleotide kinase 10 U/l (Gibco, Cat. No. 1800401)
4190 µl Milli-Q water
5 ml in total

This mix is incubated for one hour at 37ºC in a heating block. It is then incubated for 10 minutes at 80ºC to eliminate remaining enzyme activity.

The phosphorylated primers are purified on a fast, desalting FPLC column, as described by the manufacturer (Pharmacia).

2 a) Determination of purity and concentration: OD260 and OD280
The OD260 and OD280 must be measured in the resulting, purified primer dilution, which typically has a volume of 7-10 ml. The following values are used to calculate the concentration from the OD260 measurement of the purified oligonucleotide:

OD260 = 1 = 50 µg/ml DS DNA
= 40 µg/ml SS DNA and RNA
= 20 µg/ml SS oligonucleotides

The purity of the DNA is calculated using the following equation: X,

The X must be between 1.8 and 2.0 to be satisfactory. The standard concentration is 5-10 ng/µl, which results in a total amount of 35-70 µg. This is sufficient to coat 350-700 NucleoLink wells or 4-7 whole plates.

3. Reagents

3 a) DNA
The oligo can be dissolved in either Milli-Q water or TE-buffer and is typically diluted to a concentration of 50 µM in TE ( General handling of oligonucleotides).

3 b) 10 kinase buffer (10 ml)

Mix:
5000 l 1 M TRIS-HCl7,6, Final concentration: 500 mM
3333 l 0.3 M MgCl2 Final concentration: 100 mM
1000 l 0.5 M DTT Final concentration: 50 mM
667 l Milli-Q water

Aliquots of 1000 µl are prepared, and can be stored at -20ºC.

3 c) 1 M Tris-HCl7.6 (100 ml)

Mix:
12.12 g Trizma HCl
Sigma, Cat. No. T-3253
2.78 g TrizmaBase
Sigma, Cat. No. T-1503
80 ml Milli-Q water

The pH is checked and, if necessary, adjusted to 7.6.
The volume is adjusted to 100 ml with Milli-Q water.

3 d) 0.3 M MgCl2 (100 ml)

Mix:
6.10 g MgCl2, 6 Milli-Q water
Riedel de-Häen, Cat. No. 31413
100 ml Milli-Q water

The reagent can be stored at room temperature.

3 e) 0.5 M DTT (20 ml)

Mix:
1.54 g Dithiothreotol (DTT)
Sigma, Cat. No. D-0632
20 ml 0.01 M Sodium acetate (pH 5.2).

The liquid is filtered through a 0.22 µm filter and aliquots of 1000 µl are prepared and stored at -20ºC.

3 f) 0.01 M Sodium acetate - pH 5.2 (500 ml)

Mix:
0.6804 g Sodium acetate, 3 H2O
Merck, Cat. No. 6267
500 ml Milli-Q water

The pH is adjusted to 5.2.

3 g) ATP

ATP*, 100 mM (100 nmol/µl) (Boehringer Mannheim, Cat. No. 1140 965)
is diluted 1:100 in Milli-Q water to give a concentration of 1 nmol/µl.

The reagent is stored at -20ºC.
*) Adenosine 5'-triphosphate.

4. Conclusion
It is possible to directly phosphorylate the liquid phase primers and covalently bind them to the surface of NucleoLink Strips. However, in most systems the solid phase primer will only perform optimally if there is a linker in the 5'-end ( DIAPOPS results as a function of a linker in the 5'-end of the solid phase primer). Therefore, direct phosphorylation of liquid phase primer is not recommended as a method for generating solid phase primers. The solid phase primer should be purchased with both a linker in the 5'-end and with a 5'-phosphorylation.