Upgrading SELEX Technology by Using Lambda Exonuclease Digestion for Single-Stranded DNA Generation

Meltem Avci-Adali,Angel Paul,Nadj Wilhelm,Gerhard Ziemer,Hans Peter Wendel

doi:10.3390/molecules15010001

Meltem Avci-Adali, Angel Paul + Show 3 more

Open Access

https://doi.org/10.3390/molecules15010001

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Journal: Molecules	Publication Date: Dec 24, 2009
Citations: 123	License type: CC BY 4.0

Affiliation: University of Tübingen

Abstract

The generation of single-stranded DNA (ssDNA) molecules plays a key role in the SELEX (Systematic Evolution of Ligands by EXponential enrichment) combinatorial chemistry process and numerous molecular biology techniques and applications, such as DNA sequencing, single-nucleotide polymorphism (SNP) analysis, DNA chips, DNA single-strand conformation polymorphism (SSCP) analysis and many other techniques. The purity and yield of ssDNA can affect the success of each application. This study compares the two ssDNA production methods, the strand separation by streptavidin-coated magnetic beads and alkaline denaturation and the lambda exonuclease digestion, in regard to the purity of generated ssDNA and the efficiency. Here, we demonstrate the considerable benefits of ssDNA production by lambda exonuclease digestion for in vitro selection of DNA aptamers. We believe that the generation of ssDNA aptamers using this method will greatly improve the success rate of SELEX experiments concerning the recovery of target-specific aptamers.

Highlights

Single-stranded DNA templates are required during the SELEX (Systematic Evolution of Ligands by EXponential enrichment) combinatorial chemistry process for ssDNA aptamer generation [1,2,3,4] and for many other molecular biology and biotechnology applications including sensors, DNA chips [5] and microarrays, pyrosequencing technology [6], detection of point mutations in DNA using the single-stranded conformation polymorphism technique (SSCP) [7], single-nucleotide polymorphism (SNP) analysis [8], solid phase DNA sequencing [9,10], and multitude of other molecular techniques
The biotinylated double stranded DNA (dsDNA) was immobilized onto the streptavidin-coated magnetic beads
A biotinylated ssDNA aptamer was used as positive control (Figure 1, lane 4)

Summary

Introduction

Single-stranded DNA (ssDNA) templates are required during the SELEX (Systematic Evolution of Ligands by EXponential enrichment) combinatorial chemistry process for ssDNA aptamer generation [1,2,3,4] and for many other molecular biology and biotechnology applications including sensors, DNA chips [5] and microarrays, pyrosequencing technology [6], detection of point mutations in DNA using the single-stranded conformation polymorphism technique (SSCP) [7], single-nucleotide polymorphism (SNP) analysis [8], solid phase DNA sequencing [9,10], and multitude of other molecular techniques. Several methods exist for the generation of ssDNA from double stranded DNA (dsDNA), including denaturing urea-polyacrylamide gel [11,12,13,14], asymmetric PCR [15,16], lambda exonuclease digestion [17,18] and magnetic separation with streptavidin-coated beads [9,19]. The strand separation by denaturing PAGE and asymmetric PCR with subsequent purification of ssDNA from non-denaturing polyacrylamide gels are not recommendable for SELEX experiments, especially in the first selection rounds in which the binding sequences are not sufficiently amplified. Lambda exonuclease is a highly processive 5 ́Æ3 ́ exodeoxyribonuclease that selectively digests the 5 ́-phosphorylated strand of dsDNA For this purpose, a 5 ́-phosphate group is introduced into one strand of dsDNA by performing PCR where only one of the two primers is 5 ́-phoshorylated. The efficiency of both ssDNA generation methods was compared

Results and Discussion

Conclusions

Methods