Effects of Different PCR Product Purification Methods on DNA Sequencing

Direct sequencing of polymerase chain reaction (PCR) products without cloning is a rapid and efficient way of sequence analysis. Prior to direct sequencing, it is necessary to purify the PCR products from excess primers, nucleotides and enzymes that could interfere with the sequencing reaction. There are several PCR product purification methods such as spin column-based purification, enzymatic purification, ethanol precipitation and gel extraction. In this study, it is aimed to evaluate the efficiency of ethanol-ammonium acetate (EtOH-NH4Ac) and polyethylene glycol (PEG) precipitation methods for purification of PCR products prior to the dye terminator cycle sequencing. A 741 bp region of the Toll-Like Receptor (TLR) 4 gene was amplified from bovine genomic DNA using PCR. After analyzing the PCR product using agarose gel electrophoresis, it was purified with one of the following methods: a) PEG precipitation, b) EtOH-NH4Ac precipitation and c) ExoSAP-IT PCR product cleanup reagent. ExoSAP-IT reagent was used as a standard PCR product cleanup protocol. Sanger sequencing of PCR samples purified with different purification methods was performed on a Beckman Coulter CEQ8800 Genetic Analysis System. The sequence data were analyzed using Sequencing Analysis software implemented within the system. The quality check and alignment of sequences were performed using BioEdit software. The sequencing results of PCR products purified with different purification methods were compared with each other. It was found that PCR products purified with both purification methods provided good-quality sequencing templates like that of purified with ExoSAP-IT reagent.