Abstract
BackgroundThe quality of chemically synthesized oligonucleotides falls with the length of the oligonucleotide, not least due to depurinations and premature termination during production. This limits the use of long oligonucleotides in assays where long high-quality oligonucleotides are needed (e.g. padlock probes). Another problem with chemically synthesized oligonucleotides is that secondary structures contained within an oligonucleotide reduce the efficiency of HPLC and/or PAGE purification. Additionally, ligation of chemically synthesized oligonucleotides is less efficient than the ligation of enzymatically produced DNA molecules.ResultsChemically synthesized oligonucleotides with hairpin structures were acquired from our standard supplier. The stem of the hairpin contained recognition sequences for the Nt. Alw I nicking enzyme and the Mly I restriction enzyme. These double stranded regions were positioned in a way to allow self-templated circularization of the oligonucleotide. Following ligation, tandem repeats of the complementary sequence of the circular oligonucleotide could be produced through rolling circle DNA synthesis. By running successive rounds of ligation, rolling circle DNA synthesis, and nicking, the original oligonucleotide could be amplified as either the (+)-strand or the (-)-strand. Alternatively, the hairpin structure could be removed by cleavage with the Mly I restriction enzyme, thereby releasing the oligonucleotide sequence contained within the hairpin structure from the hairpin.ConclusionWe present here a method for the enzymatic production through DNA amplification of oligonucleotides with freely designable 5'-ends and 3'-ends, using hairpin-containing self-templating oligonucleotides. The hairpin comprises recognition sequences for a nicking enzyme and a restriction enzyme. The oligonucleotides are amplified by successive rounds of ligation, rolling circle DNA synthesis and nicking. Furthermore, the hairpin can be removed by cleavage with the Mly I restriction enzyme. We have named such hairpin structures "suicide cassettes".
Highlights
The quality of chemically synthesized oligonucleotides falls with the length of the oligonucleotide, not least due to depurinations and premature termination during production
Outline of the suicide cassette system An oligonucleotide able to fold into a hairpin structure by self-templated hybridization, thereby bringing the 5'-end and the 3'-end into proximity, can be circularized by the addition of a ligase [14] (Figure 1A–B)
A second round of ligation and rolling circle DNA synthesis can again be followed by a nicking reaction producing oligonucleotides with the same polarity as the one purchased (Figure 1G– H and 1J)
Summary
The quality of chemically synthesized oligonucleotides falls with the length of the oligonucleotide, not least due to depurinations and premature termination during production. This limits the use of long oligonucleotides in assays where long high-quality oligonucleotides are needed (e.g. padlock probes) Another problem with chemically synthesized oligonucleotides is that secondary structures contained within an oligonucleotide reduce the efficiency of HPLC and/or PAGE purification. Single molecule detection has become an achievable goal with the development of new techniques during recent years and we have ventured into this field ourselves [1] Some of these techniques use long chemically synthesized oligonucleotides (70–100 nucleotides) as part of the reac-. The ligation step is able to discriminate even small sequence variations in the genome [1] These circular molecules can be detected by e.g. rolling circle DNA synthesis, where long single stranded DNA molecules comprising tandem repeats of the complementary strand of the templating circle are synthesized [3,4]. With the reliance on naturally occurring restriction sites in the oligonucleotide sequence, and on additional oligonucleotides for ligation and cleavage, C2CA is more useful for signal amplification than for production of defined oligonucleotides
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