Comparison of different methods for generation of single-stranded DNA for SELEX processes.
Single-stranded DNA (ssDNA) generation is a crucial step in several molecular biology applications, such as sequencing or DNA chip and microarray technology. Molecules of ssDNA also play a key role in the selection of ssDNA aptamers through Systematic Evolution of Ligands by EXponential enrichment (SELEX). With particular interest for this application, herein we present a comparative study of the most used methods for generation of ssDNA used in SELEX, such as asymmetric PCR, enzyme digestion and magnetic separation with streptavidin beads. In addition, we evaluate a new technique that combines asymmetric PCR and enzyme digestion with the aim to achieve the maximum efficiency in ssDNA generation. The methods studied were compared in terms of quality of ssDNA using electrophoretic analysis and generated ssDNA yields were quantitatively measured using an Enzyme-Linked OligoNucleotide Assay (ELONA).
Nanobiotechnology and Bioanalysis group, Department of Chemical Engineering, Universitat Rovira i Virgili, Tarragona, Spain.
M
M
Svobodová
A
A
Pinto
P
P
Nadal
C K
CK
O' Sullivan
D052157:Aptamers, Nucleotide / Q000737:chemistry; D001710:Biotin / Q000737:chemistry; D005591:Chemical Fractionation / Q000379:methods*; D017931:DNA Primers / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry*; D004587:Electrophoresis, Agar Gel; D005090:Exodeoxyribonucleases / Q000737:chemistry; D015723:Gene Library; D006735:Horseradish Peroxidase / Q000737:chemistry; D059346:Magnets; D016133:Polymerase Chain Reaction / Q000379:methods*; D052156:SELEX Aptamer Technique / Q000379:methods*; D019809:Streptavidin / Q000737:chemistry
D052157:Aptamers, Nucleotide; D017931:DNA Primers; D004277:DNA, Single-Stranded; D001710:Biotin; D019809:Streptavidin; D006735:Horseradish Peroxidase; D005090:Exodeoxyribonucleases