Site‐Selective Blocking of PCR by a Caged Nucleotide Leading to Direct Creation of Desired Sticky Ends in The Products

Abstract

In order to terminate the polymerase reaction at a desired position, a caged thymine derivative--4-O-[2-(2-nitrophenyl)propyl]thymine--was incorporated into PCR primers. In the PCR cycles, the elongation of the nascent strand (5'-->3' direction) by polymerase was site-selectively terminated at the 3'-side of T(NPP). Accordingly, predetermined protruding ends were obtained after the removal of the protecting group by short UVA irradiation. Recombinant vectors coding the GFP gene were successfully prepared by direct ligation of these light-assisted cohesive-ending PCR (LACE-PCR) products with scission fragments obtained by use either of restriction enzymes or of artificial restriction DNA cutters and were used for transformation of E. coli.

Turning the 10–23 DNAzyme On and Off with Light

“…Photoactivatible (also known as ‘caged’) oligonucleotides have been used primarily to study gene expression,[10] and more recently, have also been used for controlling PCR product generation,[11] RNA folding,[12] oligonucleotide mismatch detection[13] and conformation switches used in nanomachines. [14] The focus of the current study is the 10–23 DNAzyme, a Mg 2+ -dependent enzyme that catalyzes RNA cleavage at a pyrimidine-purine junction, preferentially AU.…”

Nucleotides and Nucleic Acids; Oligo- and Polynucleotides

“…184,185 The same protecting group has also been attached to O4 of thymidine, with similar photoactivated control of PCR. 186 Oligonucleotides bearing N3-azidoethyl and a N3-propargyl modifications at the termini have been used to prepare cyclic DNA via a triazole linkage. 41 The most common uracil O4 modification is 4-thiouracil, which is isoelectronic with uracil, but undergoes photolytic crosslinking reactions.…”

Cut-and-Paste of DNA Using an Artificial Restriction DNA Cutter

“…When necessary, DNA fragments that can be directly ligated with ARCUT product (without the use of “jointing oligonucleotide”) can be prepared by use of “light-assisted cohesive-ending PCR” (LACE-PCR) [17]. In order to form a terminus in which one strand is protruding the other by 10–20 nucleotides like ARCUT product, photo-caged nucleotides are incorporated to the primers used for the PCR.…”