- Methodology article
- Open Access
A new enzymatic route for production of long 5'-phosphorylated oligonucleotides using suicide cassettes and rolling circle DNA synthesis
© Lohmann et al; licensee BioMed Central Ltd. 2007
- Received: 28 March 2007
- Accepted: 16 August 2007
- Published: 16 August 2007
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. Additionally, ligation of chemically synthesized oligonucleotides is less efficient than the ligation of enzymatically produced DNA molecules.
Chemically 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.
We 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".
- Wash Buffer
- Hairpin Structure
- Rolling Circle
- Successive Round
- Single Molecule Detection
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 . Some of these techniques use long chemically synthesized oligonucleotides (70–100 nucleotides) as part of the reaction set-up, e.g. padlock probes . A padlock probe is a single stranded oligonucleotide which upon correct hybridization to a target sequence has its 5'-end and 3'-end brought into immediate proximity, allowing for circularization of the padlock probe by ligation. The ligation step is able to discriminate even small sequence variations in the genome . 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]. Padlock probes in combination with rolling circle DNA synthesis have been used for the in situ detection of DNA [1, 5], microarray-based detection of viruses  and for SNP detection in combination with PCR [7, 8]. Reliably achieving single molecule detection requires perfection in every step of the reaction, including perfect reagents, such as flawless oligonucleotides. A problem when using long chemically synthesized oligonucleotides is that during chemical synthesis the coupling efficiency is approximately 99% per nucleotide. For an oligonucleotide consisting of 25 nucleotides the coupling efficiency can therefore be estimated to be (0.99)24 = 0.79 (24 couplings), whereas an oligonucleotide consisting of 100 nucleotides achieves an estimated coupling efficiency of (0.99)99 = 0.37 (99 couplings). Furthermore, the occurrence of depurination and nucleotide skipping increases with the length of the oligonucleotide [9, 10]. Thus, not only the yield but also the quality of long chemically synthesized oligonucleotides may become unsatisfactory. Some of these problems can be reduced by using one or more purification steps (e.g. PAGE and/or HPLC). However, if secondary structures are present in the oligonucleotide, high-quality purification is difficult. Numerous techniques already exist for DNA amplification of oligonucleotides through enzymatic synthesis, such as cloning , PCR  and rolling circle DNA synthesis. The latter method, the so-called circle-to-circle amplification (C2CA) , is a method for the amplification of a single stranded DNA sequence, based on successive rounds of ligation, rolling circle DNA synthesis and restriction digestion. Rolling circle DNA synthesis has the advantage over PCR that the multiple copies are made directly from the original circle and not as copies of copies. However, 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.
We now present a novel rolling circle method for the enzymatic production of long 5'-phosphorylated oligonucleotides with improved ligation efficiencies compared to chemically synthesized oligonucleotides and with freely designable 5'-ends and 3'-ends. This is achieved by the inclusion of a hairpin structure (termed suicide cassette) comprising binding and cleavage sites for both nicking and restriction enzymes into the starting oligonucleotide.
Outline of the suicide cassette system
Leaving the suicide cassette on the oligonucleotide provides a new class of circle probes, which, due to their ability to self-circularization, may have advantages for the detection of RNA (Stougaard et al., manuscript under revision).
Detailed description of the suicide cassette
Amplification of an oligonucleotide contained within a suicide cassette
Solid support amplification to verify purity and ligation of the enzymatically synthesized oligonucleotides
We present a novel method for the production of 5'-phosphorylated oligonucleotides amplifying a hairpin-containing oligonucleotide through rolling circle DNA synthesis. The hairpin is equipped with binding and cleavage sites for a nicking enzyme, which enables nicking and ligation without the addition of a supplementary oligonucleotide. Furthermore, the hairpin contains binding and cleavage sites for the Mly I restriction enzyme, enabling removal of the complete hairpin from the rest of the oligonucleotide. This method can be used for production of high quality 5'-P-oligonucleotides with superior ligation efficiencies.
5'-P-CTGCCATCTT AACAAACCCT CGACCTCAAT GCTGCTGCTG TACTACTCTT ATGCGATTAC CGGGCT
5'-P-GTCGATCCCT GCCATCTTAA CAAACCCTCG ACCTCAATGC TGCTGCTGTA CTACTCTTAT GCGATTACCG GGCTGGATCG ACTCGGAATT TCTTCCGA-3'
Pr SF-WT90 (+)
5'-GTAGTACAGC AGCAGCATTG AGG-3'
Pr SF-WT90 (-)
5'-CCTCAATGCT GCTGCTGTAC TAC-3'
Amin-L16-Pr SF-WT90 (+)
5'-AMIN-CCTTCCTTCC TTCCTTGTAG TACAGCAGCA GCATTGAGG-3'
Amin-L16-Pr SF-WT90 (-)
5'-AMIN-CCTTCCTTCC TTCCTTCCTC AATGCTGCTG CTGTACTAC-3'
Amin-L16-Mly I (+)
5'-AMIN-CCTTCCTTCC TTCCTTTTGT TAAGATGGCA GAGCCCGGTA ATCGCA-3'
Amin-L16-Mly I (-)
5'-AMIN-CCTTCCTTCC TTCCTTTGCG ATTACCGGGC TCTGCCATCT TAACAA-3'
5'-TAMRA-CCTCAATGCT GCTGCTGTAC TAC-3'
Anti ID 16
5'-TAMRA-GTAGTACAGC AGCAGCATTG AGG-3'
Amplification of DNA oligonucleotides
5'-phosphorylated oligonucleotides were ligated with T4 DNA ligase (Fermentas, Vilnius, Lithuania). Rolling circle DNA synthesis was performed for 16 hours at 37°C in a mixture containing 1× Phi29 DNA polymerase buffer (Fermentas), 1.5 nM ligated oligonucleotide, 1.5 nM primer, 250 μM dNTP, and 0.25 u/μl Phi29 DNA polymerase (Fermentas). The reactions were stopped by heat inactivation for 10 minutes at 65°C. Nicking was performed by the addition of 1 volume of a nicking mixture containing 1× NEBuffer 2 (NEB, Ipswich, MA, USA) and 1 u/μl of Nt. Alw I (NEB). Nicking reactions were incubated for 16 hours at 37°C and stopped by heat inactivation for 20 minutes at 80°C. The amount of cleavage products can be estimated by PAGE. Ligation reactions were performed by the addition of ATP and ligase to the inactivated reaction mixtures. The second round of rolling circle DNA synthesis was performed as the first one using a primer complementary to the one used in the first round. Further rounds of amplification can be performed as described above. To remove the suicide cassette, thereby terminating the amplification, cleavage of the rolling circle product was done by the addition of 1 volume of cleavage mixture containing 1× NEBuffer 4 (NEB), 0.2 μg/μl BSA (NEB) and 1 u/μl of Mly I (NEB). Cleavage reactions were incubated for 16 hours at 37°C and stopped by heat inactivation for 20 minutes at 65°C. Products can be separated and purified by PAGE.
Polyacrylamid gel electrophoresis (PAGE)
Amplified and cleaved products could be separated by PAGE. Products were separated on 10% denaturating gels and stained with SYBR Gold. Images were visualized using a Gel Doc 1000 (Biorad).
When indicated the cleaved products were purified from the gel using standard phenol/chloroform purification and the concentration estimated by photospectrometry.
Solid support amplification and detection of Nt. Alw I and Mly I cleaved rolling circle products
5'-amine coupled primers were linked to CodeLink Activated Slides (GE Healthcare) according to the manufacturers protocol. Detection after nicking: Nicked products (actual concentrations in individual experiments are given in the figure legends) were ligated in 1× ligation buffer (Fermentas), inactivated, supplemented with 0.5 M NaCl (final concentration) and hybridized to the solid support for 30 minutes at 37°C in a humidity chamber. Detection after Mly I cleavage: Cleaved products (actual concentrations in individual experiments are given in the figure legends) were ligated directly onto the coupled primes in a mixture containing 1× ligase buffer (Fermentas), 0.2 μg/μl BSA, 250 mM NaCl, and 0.05 u/μl T4 DNA ligase (Fermentas) for 30 minutes at 37°C in a humidity chamber. The slides were washed in 0.1 M Tris-HCL, 150 mM NaCl and 0.3% SDS (wash buffer 1) for 2 min at room temperature followed by a wash in 0.1 M Tris-HCL, 150 mM NaCl and 0.05% Tween-20 (wash buffer 2). Rolling circle DNA synthesis was performed for 30 minutes at 37°C in 1× Phi29 buffer, 0.2 μg/μl BSA, 250 μM dNTP, 5% glycerol, and 1u/μl Phi29 DNA polymerase. The reactions were terminated by washing 2 minutes in wash buffer 1 and 2 minutes in wash buffer 2. The rolling circle products were detected by hybridizing fluorescently labeled oligonucleotides in a buffer containing 20% formamide, 2× SSC, 5% glycerol and 0.17 μM of either ID 16 or anti ID 16 for 30 min at 37°C. Slides were washed for 2 min at room temperature in wash buffer 1 and wash buffer 2 and dehydrated through a series of ethanol and mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA).
Solid support assays were analyzed with an epifluorescence microscope (Leica, Wetzlar, Germany) and images were recorded with a SenSys CCD-camera operated by the SmartCapture 2 version 2.0 from Digitalscientific (Cambridge, UK). A 63× objective (Leica) was used for all images. Thresholding was performed using Adobe Photoshop (Adobe Systems).
This project was funded by the Moltools program under EU-framework 6, the John and Birthe Meyer foundation, Arvid Nilssons Fond, Aase og Ejnar Danielsens Fond and Civilingeniør Frode V. Nyegaard og hustrus Fond. The authors wish to thank Stephen Hamilton-Dutoit for language revision of the manuscript.
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