Standard oligos
General Overview Oligo Synthesis
Biolegio uses optimized phosphoramidite chemistry and reagents of the highest quality. Order handling and oligo synthesis is performed on a completely integrated computer platform. Our home built synthesis programs result in a typical coupling efficiency of 99%. With this efficiency we are able to synthesize oligos with lengths up to 125 bases. Synthesis is performed under salt free conditions, which avoids the need for additional purification for most basic molecular biology applications, such as PCR, RT-PCR, sequencing, hybridization studies, and antisense studies. Additional purification by HPLC, PAGE or cartridge is available for more sensitive applications.
DNA synthesis is performed from 3’ to 5’. Bases are attached to the growing chain according to the standard ß-cyanoethyl chemical reactions. Each cycle consists of:
- Deblocking: the first nucleotide, attached to the solid support (CPG or polystyrene) is deprotected by removing the DMT-protecting group. The result is a free 5’- hydroxyl group which is able to react with the next nucleotide.
- Coupling: the next nucleotide is added to the reaction and is covalently attached (i.e. coupled) to the previous nucleotide.
- Capping: any of the first nucleotide that failed to react is capped so that it will no longer participate at any subsequent step.
- Oxidation: the bond between the first nucleotide and the successfully coupled second nucleotide is oxidized to stabilize the growing chain.
- Deblocking: the 5’ DMT group is removed from the second nucleotide to prepare it for further cycles.
In addition of our standard oligos, Biolegio offers an extensive series of available modifications. Some are described on this site. Please contact Biolegio if you do not find information about the modification you would like.
Quality Control
A stringent quality control system ensures that you can expect the quality of our oligonucleotides to be of the highest standard. Before the synthesis, all chemicals are checked to meet our quality standard. During the fully automatic oligo synthesis, all steps of the synthesis are monitored by multiple control functions on our DNA synthesizers. This way we can assure the coupling efficiency of the synthesis to meet our demands.
After synthesis and deprotection of the oligo, they are checked further to guarantee that they meet our quality standard. All oligos are routinely analyzed by optical density (OD) measurement. In addition, oligos are randomly analyzed by gel electrophoresis. Any oligo that fails to meet our specifications is resynthesized. The result of our stringent QC process is a high-quality oligonucleotide.
All oligos are delivered with our Products Specifications Sheet that includes: sequence, yield in nmol and µgram, melting temperature and MW.
Synthesis Scales and Yield
Biolegio offers four different synthesis scales: 10 nmol,40 nmol, 200 nmol and 1000 nmol. For nonlabeled, standard oligos, up to 30 bases, we guarantee a minimum yield:
- 10 nmol scale: 4,5 nmoles
- 40 nmol scale: 20 nmoles
- 200 nmol scale: 95 nmoles
- 1000 nmol scale: 400 nmoles
Furthermore, you have the ability to chose between two different kinds of synthesis protocols, dependant on the application,length and features of the oligo.
Economy synthesis method
This method uses relatively long periods for the different couplingsteps. This method is particular usefull for synthesis of short (< 30 bases) oligonucleotides.
High Purity synthesis method
The quality of oligos synthesized with this method will be even higher than the ones synthesized with the Economy method. This method is particular recommended for longer (> 30 bases) and modified oligonucleotides.
Available lengths vs. Scales
The different scales have restrictions regarding the length of the oligo that can be synthesized.
- 10 nmol scale: up to 40 bases
- 40 nmol scale: up to 70 bases
- 200 nmol scale: up to 90 bases*
- 1000 nmol scale: up to 150 bases*
(* For long oligo’s it is recommended for some applications to have them purified. See section Purification)
Shipment
The oligos can be shipped dry or dissolved in a requested solution. They can be packed in individual-labeled microcentrifuge tubes, or in 96 wellsplate-format.
Under normal conditions standard oligos are shipped within one day. Shipment of larger orders, purified oligos and labeled oligos will take a few days more. Please contact Biolegio for information about delivery times and shipment costs when using express mail services.
Modified oligonucleotides
Incorporation of a modification into an oligonucleotide
Oligos can be modified in several different ways by utilizing the active groups of the nucleotide or creating nucleotide analogues. Modifications can occur either during (direct) or after synthesis (indirect).
- Direct incorporation of modified nucleosides during automated DNA synthesis. Using this method, modified bases can be incorporated internally or on the 5’ end. Thymidine analogues (which replace T-bases in the sequence) are frequently used as modified bases. This method is site-specific but is constrained by the availability of specialized phosphoramidites.
- 3’ end modification using special solid support. Once again, this method relies on the existence of modified CPG columns.
- Modifications using functional groups (via an amino modifier, via the 3’ or 5’ hydroxyl groups, phosphate group or thiollinker). Although the yields are often low, the reaction of functional groups attached to the bases with activated dyes molecules is widely used to label oligos either on the ends or internally.
Degenerated oligos
Standard "wobbles" are mixtures of two or more different bases at a given position within the sequence. Wobbles are often incorporated into oligonucleotide probes designed to hybridize to an unknown gene that encodes a known amino acid sequence. Since the genetic code is degenerate, the probe is prepared with wobbles at the degenerate sites.
The use of mixed base additions in the design of a degenerated oligo is best if the number of degenerate sites is small. The wobbles, except for the ones at the 3’-site are synthesized with no extra costs.
The standard code letters for specifying a wobble are:
R= A/G Y= C/T
M= A/C K= G/T
S= C/G W= A/T
B= C/G/T D= A/G/T
H= A/C/T V= A/C/G
N= A/C/G/T I= Inosin