title   
  

Characterization of a dual biotin tag for improved single stranded dna production

Yüce, Meral and Kurt, Hasan and Budak, Hikmet (2014) Characterization of a dual biotin tag for improved single stranded dna production. Analytical Methods, 6 (2). pp. 548-557. ISSN 1759-9660 (Print) 1759-9679 (Online)

This is the latest version of this item.

[img]PDF - Registered users only - Requires a PDF viewer such as GSview, Xpdf or Adobe Acrobat Reader
892Kb

Official URL: http://dx.doi.org/10.1039/c3ay41899e

Abstract

Generation of single-stranded DNA plays a key role in many biotechnology applications including production of aptamers, single strand conformation polymorphism, nuclease S1 mapping, pyrosequencing, genosensors, probe preparation and labelling, subtractive hybridization as well as nucleic acid sensing and microarrays. Several methods are available in the literature to produce single-stranded DNA from double-stranded DNA template, such as extraction of the sense strand from denaturing gels, asymmetric PCR, use of streptavidin-biotin interaction, and some alternative methods, including enzymatic digestion of negative strand by either Lambda exonuclease or T7 Gene 6 exonuclease. In this report, a detailed characterization of dual biotin tag method to generate single-stranded DNA from the random oligonucleotide library is presented. Unlike the traditional streptavidin-biotin method that uses single biotin tagged molecule during separation, this novel technique is based on a dual biotin molecule covalently attached to 5’ end of the negative strand. Improved technique takes less than one hour as a consequence of eliminated alkali treatment step, which make this procedure the shortest procedure described in literature so far for single-stranded DNA production. The method can achieve a single-stranded DNA yield around 75% from the corresponding DNA template in Tris-HCl buffer. A number of parameters, such as the effect of different elution buffers and heat treatments, spontaneous release of streptavidin from the magnetic bead surface, loss of beads during consecutive washes, aggregation of the beads, were investigated to reveal the optimal conditions for single-stranded DNA production. FTIR, DLS, SEM, and Electrophoresis techniques were used for characterization studies.

Item Type:Article
Uncontrolled Keywords:Dual Biotin, single-stranded DNA, Aptamer, Oligonucleotide, Magnetic beads
Subjects:Q Science > Q Science (General)
ID Code:24609
Deposited By:Meral Yüce
Deposited On:10 Dec 2014 15:38
Last Modified:10 Dec 2014 15:38

Available Versions of this Item

  • Characterization of a dual biotin tag for improved single stranded dna production. (deposited 10 Dec 2014 15:38) [Currently Displayed]

Repository Staff Only: item control page