SSCP Screening of Individual Aptamers

Abstract

Aptamers are specific binding nucleic acids that emerge from in vitro selection. During the systematic evolution of ligands by exponential enrichment (SELEX) procedure, analysis of the sequences of the numerous selected individual molecules becomes an important step in the final stage of aptamer selection. The sequencing of cloned aptamers from the selected pool generally reveals groups of identical sequences and rarely occurring individual aptamers. This study demonstrates an approach similar to the single strand conformation polymorphism (SSCP) method used for mutation testing in genes. Human angiotensin I-specific aptamers have been used to show the efficiency of the SSCP method to classify selected individual sequences into identity groups, which minimizes sequencing efforts. Additionally this approach allows the rapid isolation and identification of aptamers from a mixture.

“Mirror” Method to Estimate Mutagenic Activity of DNA Lesions

“…To find a suitable restriction enzyme for the analysis of a particular DNA lesion, it is a prerequisite to determine which mutations that lesion causes. Detecting and identifying its mutations can be achieved by the SSCP method [15]. It is based on the fact that different single-strand DNA molecules, depending on their nucleotide sequence, differ in their electrophoretic mobility through the non-denaturing polyacrylamide gel.…”

“…In the past, we successfully employed it in the identification and separation of single-strand DNA aptamers of equal length, differing in their nucleotide sequence. Even scarcely present nucleotide sequences can be isolated for further amplification and sequencing [15]. The same procedure can be performed with mutant DNA molecules obtained after the DNA synthesis past various damaged sites, making the study of lesions other than 8-oxoG possible.…”

RNA aptamers selected against DNA polymerase inhibit the polymerase activities of DNA polymerases and

“…Individual RNA aptamers were synthesized by in vitro transcription from PCR products obtained by the direct amplification of bacterial lysates ( 25 ). In vitro transcription reactions were performed in a mixture containing: 40 mM Tris–HCl (pH 7.9), 25 mM MgCl 2 , 30 mM DTT, 2 mM each rNTP, 40 U of RNAse inhibitor (Roche Applied Science, Indianapolis, IN, USA), 200 U of T7 RNA polymerase (Stratagene, La Jolla, CA, USA) and 70 pmol of purified (PCR Cleanup Kit, Qiagen, Valencia, CA, USA) PCR product.…”