DMTO[ODNo opo[ODN oh

Scheme 5. On-column synthesis of ferrocene-labeled oligodeoxynucleotides. Reagents and conditions: (a) DNA synthesis; (b) off-synthesizer, Pd(Ph3P)4 Cul, TEA, DMF, 3 hr; (c) resume DNA synthesis; (d) 30% NH4OH, 55°, 16 hr; ODA, Oligodeoxynucleotide; Prot., protected.

material undergoes adsorption and desorption processes between the nonpolar stationary phase and polar mobile phase. The hydrophobic interactions between the oligodeoxynucleotides and the stationary or mobile phase determine the retention time. In most cases, functionalized oligodeoxynucleotides are more hydrophobic and thus have a longer retention time than unlabeled oligodeoxynucleotides. Besides purification, HPLC also provides a means to determine the yield.

A typical purification procedure for a labeled oligodeoxynucleotide involves the following steps.

1. Deprotect the synthetic oligodeoxynucleotide (1 /zmol) after DNA synthesis with NH4OH at 55° for 12 hr.

2. Lyophilize the sample and redissolve the residue in 100 /¿l of deionized water.

3. Equilibrate the reversed-phase Qg column (semipreparative) with 95% TEAA buffer and 5% acetonitrile.

4. Inject 20-30 /il of sample and elute with a gradient of 5-50% acetonitrile over 30-40 min.

5. Collect the desired product as it elutes from the column by monitoring the oligodeoxynucleotide absorbance at 254 nm.

Polyacrylamide Gel Electrophoresis

Polyacrylamide gel electrophoresis (PAGE) is routinely used to separate and purify synthetic oligodeoxynucleotides.66 DNA possesses a negative phosphate backbone and consequently orients and migrates in an electric field. The elec-trophoretic mobility of single-stranded or double-stranded DNA is closely related to its chain length. The larger the DNA, the slower it migrates. The redox-labeled oligodeoxynucleotide typically moves more slowly compared with an unlabeled oligodeoxynucleotide. The effective range of separation depends on the different concentrations of polyacrylamide gel. For example, a denaturing 20% acrylamide gel containing 7-8 M urea is able to separate oligodeoxynucleotides whose lengths differ by as little as 0.2% (1 in 500 bases).

A typical procedure is outlined below.

1. Deprotect the synthetic oligodeoxynucleotide (1 /xmol) after DNA synthesis with NH4OH at 55° for 12 hr.

2. Lyophilize the sample and redissolve the residue in a solution of 20 ¡i\ of deionized water and 40 ¡jlI of loading buffer (95% formamide, 20 mAf EDTA, 0.25% xylene cyanol).

66 J. Sambrook, E. F. Fritsch, and T. Maniatis (eds.), "Molecular Cloning: A Laboratory Manual," 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.

3. Prepare a 20% denaturing polyacrylamide gel (15 ml of 40% acrylamide-bisacrylamide, 3 ml of lOx TBE, 14.5 g of urea).

4. Apply 5 /xl of the sample to each well of the gel and run the gel for 2-3 hr.

5. Visualize the bands under UV light by laying the gel (covered with Saran Wrap) on TLC plates.

6. Slice the desired band, and crush and elute the desired oligodeoxynucleotide with sodium acetate buffer.

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