( Jolanta Kolodziejek MSc...)Comparison of PCR Kleen Spin Columns to Traditional Methods for Purification of PCR Products Prior to Sequencing, Rev A

Jolanta Kolodziejek, MSc, and Professor Norbert Nowotny, PhD, Clinical Virology Group, Institute of Virology, University of Veterinary Medicine, Vienna , Veterinaerplatz 1, A 1210 Vienna, Austria

Introduction
The purity of the DNA template employed in sequencing PCR (cycle sequencing) is the most critical factor in nucleotide sequencing. The presence of contaminants such as enzymes (nucleases, polymerases), salts, primers, primer- dimers , nucleotides (dNTPs or ddNTPs), ethanol, and others decre ases the quality of the sequencing scans significantly. Primer- or RNA-contaminated samples exhibit a high background and lead to misinterpretation of sequencing data. Contamination with nucleases, salts, nucleotides, and ethanol decreases read length and signal intensity and sometimes results in unre a dable sequence data.

We tested Bio-Rads PCR Kleen spin columns for purification of PCR products prior to sequencing PCR in two different experiments, and compared the resulting nucleotide sequences obtained from these samples with the sequences obtained after purification by traditional methods.

Methods
Experiment 1. Comparison of Effectiveness of Purification Method Prior to Sequencing PCR
A 510 bp RT-PCR product (100 l) was prepared using Borna disease virus as template, and three of four equal-volume aliquots of the PCR product were purified by different methods prior to sequencing. The fourth aliquot was left as an unpurified control.

The first aliquot of the PCR product was purified using PCR Kleen spin columns according to the instructions. The DNA f rom the other two aliquots was recovered from agarose sli
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