Effects of Different PCR Product Purification Methods on DNA Sequencing

Year 2025, Volume: 25 Issue: 2, 234 - 239

Diğdem Aktopraklıgil Aksu

Abstract

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.

Keywords

DNA sequencing, PCR product, PEG, EtOH-NH4Ac, ExoSAP-IT, CEQ8800 genetic analysis system

Ethical Statement

The author declares that she complies with all ethical standards.

Supporting Institution

Prof. Alessio Valentini and Dr. Lorraine Pariset, University of Tuscia, Italy

Thanks

I would like to thank Prof. Alessio Valentini and Dr. Lorraine Pariset, University of Tuscia, Italy for providing me laboratory facilities and funding for this study.

Farklı PCR Ürünü Saflaştırma Yöntemlerinin DNA Dizilemeye Etkisi

Abstract

Polimeraz zincir reaksiyonu (PCR) ürünlerinin klonlama olmadan doğrudan dizilenmesi, dizi analizinin hızlı ve etkili bir yoludur. Doğrudan dizilemeden önce PCR ürünlerinin, dizileme reaksiyonuna müdahale edebilecek fazla primerlerden, nükleotidlerden ve enzimlerden arındırılması gerekir. Spin-kolon bazlı saflaştırma, enzimatik saflaştırma, etanol çöktürmesi ve jel ekstraksiyonu gibi çeşitli PCR ürün saflaştırma yöntemleri vardır.
Bu çalışmada PCR ürünlerinin boya sonlandırıcı döngü dizilemesi öncesinde saflaştırılmasında etanol-amonyum asetat (EtOH-NH4Ac) ve polietilen glikol (PEG) çöktürme yöntemlerinin etkinliğinin değerlendirilmesi amaçlanmıştır. Toll-benzeri reseptör (TLR) 4 geninin 741 bç'lik bölgesi, sığır genomik DNA'sından PCR ile çoğaltılmıştır. PCR ürünü agaroz jel elektroforezi kullanılarak analiz edildikten sonra aşağıdaki yöntemlerden biri ile saflaştırılmıştır: a) PEG çöktürmesi, b) EtOH-NH4Ac çöktürmesi ve c) ExoSAP-IT PCR ürünü temizleme reaktifi. ExoSAP-IT reaktifi standart PCR ürünü temizleme yöntemi olarak kullanılmıştır. Farklı saflaştırma yöntemleriyle saflaştırılan PCR örneklerinin Sanger dizi analizi Beckman Coulter CEQ8800 Genetik Analiz Sistemi ile gerçekleştirilmiştir. Dizi verileri sistemde yer alan Dizi Analizi yazılımı kullanılarak analiz edilmiştir. Dizilerin kalite kontrolü ve hizalanması BioEdit yazılımı ile gerçekleştirilmiştir. Farklı saflaştırma yöntemleriyle saflaştırılan PCR ürünlerinin dizileme sonuçları birbirleriyle karşılaştırılmıştır. Her iki saflaştırma yöntemiyle saflaştırılan PCR ürünlerinin ExoSAP-IT reaktifi ile saflaştırılana benzer şekilde iyi kalitede dizileme kalıbı sağladığı bulunmuştur.

Keywords

DNA dizileme, PCR ürünü, PEG, EtOH-NH4Ac, ExoSAP-IT, CEQ8800 genetik analiz ssitemi

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