|Storage of oligo-nucleotides||General information on purity||Primer that does not work||Non-standard bases||Non-standard linkages|
Oligos are shipped in 250µl 50 mM Tris - 2 mM EDTA pH 7.5. The OD value is recorded on the vial. Oligos should be aliquoted into sterile vials with one stored at 4ºC and the remainder at -20ºC or better still, -70ºC. Lyophylization may be another good option. Quantification of primers is based on the average residual mass of a base (305 D) and the average absorptivity. 1 OD unit is equivalent to 33 µg. A sample of 20 mer containing 100 OD units/ml thus has a MW = 20 × 305 = 6100 daltons and a concentration of 3.3 mg/ml or 0.54 µmoles/ml or 0.54 mM (accuracy ~ 5%).
For more accurate calculations the actual MW and molar absorption coefficient based on the composition can be calculated.
MW = A × 312.2 + G × 328.2 + C × 288.2 + T × 303.2 - 61
E254 = (A × 15.4 + G × 11.7 + C × 7.3 + T × 8.8) × 0.9
A, G, C and T are the number of each base in the primer, E is the micromolar extinction coefficient and 0.9 a correction factor for base stacking. The primer can deteriorate on standing due to secondary structure formation. A brief exposure to 100ºC followed by snap cooling (e.g. dry ice) may solve this problem. Deterioration can also be due to various other reasons not yet well understood. Olinucleotides are single stranded and care should be taken to avoid nuclease contamination. (EDTA is a nuclease inhibitor and gives some protection.)
The average coupling efficiency in our phosphoramidite synthesis method is above 99% and it is therefore unnecessary to purify our primer for a large number of applications. All synthetic oligonucleotides contain closely related, but slightly truncated, failure sequences. Typically, an instrument that attains a 99% coupling efficiency would yield 83% of a specified 20-mer and 50% of a 70-mer. The failure sequences range from (the specified 20-mer) a 19-mer down to a family of shorter sequences in continuously decreasing amounts. This phenomenon is part-and-parcel of the process and universally observed. Coupling yields may however vary from lab to lab (98% - 99.5%)
Typical failure sequences for a high yielding 17mer (10K)
The detritylation value for each nucleotide given at the end of the enclosed "Synthesis Document" reflects this repetitive yield. (Synthesis proceeds from the 3' to the 5' end and the 5' end is protected by a trityl group (DMTrO) which is removed prior to the addition of the next nucleotide which is attached to the 5' OH group). Oligos are detached from the synthesis resins with 33% NH4OH followed by deprotection at 70ºC for 9mins. The NH4OH is evaporated followed by a butanol precipitation, redissolved in the TRIS-EDTA buffer and quantitated by spectroscopy (OD at 260nm).
Should you require very pure primers one or another purification must be considered.
- Polyacrylamide electrophoresis followed by excision and extraction of the major band.
(Eckstein: Oligonucleotides and Analogues)
NOTE : Failure sequences may not always be apparent as they are present in smaller amounts and are present as many different and smaller molecular weight bands.
- Oligonucleotides can be purified on reverse phase cartridge in a batch type process. This is best done while the trityl group is still attached to the polymer. The trityl group is removed with 80% acetic acid followed by another ethanol precipitation after the cartridge purification.
- HPLC on a reverse phase column, using TEAA- acetonitrile buffer with the trityl group still attached to the 5' end (TRITYL-ON). The trityl- group is removed afterwards with acetic acid.
- HPLC on an ion-exchange column using a salt gradient to elute the oligonucleotides. This is the most powerful of all four methods.
From time to time we encounter primer that does not work. The reason for this can be manyfold. Incorrect primer. With each primer we supply a synthesis report which gives information on the sequence and the progress of the synthesis in terms of tritylation values. These values give an indication of success of the synthesis. It is possible to estimate graphically the repetitive yield which in most cases is in the order of 99.5%. Secondly the quality of the product can be determined by subjecting the olinucleotide to polyacrylamide electrophoresis under denaturing conditions. Both the size and the presence of additional bands will reveal the quality of the product. A correct size and a small amount of failure sequences all travelling faster than the full length product indicate a good product. Secondary structure. Complementary sequences to itself or other primers give rise secondary structures and other problems which invariably results in failed PCR experiments. Remedy - correct primer design. Other important aspects in primer design are overall GC content (50%) and using a C or G clamp. Unfortunately other problems can arise. Depurination of primer is not easily detected as the electrophoretic mobility is only slightly affected. If we suspect problems of this nature we subject the primer to composition analysis after hydrolysis. Incorrect PCR conditions is another possibility. These have to be eliminated by incorporating appropriate blanks and controls. We are however fairly sympathetic to problems and should it be established that the fault lay with us, we will refund your money and supply you a free primer of your choice.
There are a number of modified bases and base analogues that we can employ in the synthesis of oligonucleotides depending upon your requirements. A number of these are outlined below. If the oligo modification you need is not listed, please call, we may expand our range of custom modifications if the demand is high enough.
These minor nucleotides can be substituted for the regular bases anywhere (except the 3'-end) in your oligonucleotide. We currently carry the following minor nucleotides:
- deoxy Inosine (dI)
- deoxy Uridine (dU)
- 5-Methyl-deoxy Cytidine (5-Me-dC)
- O-6-Methyl-deoxy Guanosine (O6-Me-dG)
S-Oligos are specific inhibitors of gene expression that are capable of forming RNase H sensitive hybrids. They are resistant to nucleases in cells and media.
BIOTINYLATES OLIGOS (Bio TEG PHOSPHORAMIDITE)
This reagent allows for multiple biotin additions throughout oligos which can be used to increase the signal of the probe in hybridization studies.
A variety of modifiers are available that modify or derivatives the 5'-end, 3'-end, or the internal structure of an oligo.
5'-AMINO MODIFIERS (Amino linker)
5'-amino modifiers that can be used to synthesize oligos with 5'-primary amino groups. The primary amines can be used as sites to which other groups can be attached, such as biotin or fluorescent labels among others. Linkers ranging in size from 3 to 12 carbons are available. At this stage we only clock the C6-TFA amino linker which is frequently used to couple fluorescent tags for use in automated fluorescent sequencers.
This modifier can be used in place of a thymidine during synthesis. The primary amine is separated from the base by a 10 atom spacer arm. After deprotection, the primary amine can be labelled or attached to an enzyme or insoluble support. Several can be added.
We offer a thiol-modifier, with a C6 S-S linker, which can be used to introduce a 5'-thiol group onto the end of an oligo. Similarly, this thiol-containing group can be used as a point to attach other thiol-reactive groups.
This is a chemical phosphorylating reagent designed to introduce chemically a 5'-phosphate group into an oligo as an alternative to phosphorylating an oligo enzymatically. This reagent can also be used for 3'-phosphorylation.
Biotin is introduced on a C12 linker chain at the 5' position. 3'-modifications are also available.
Digoxigenin (DIG) is a synthetic steroid hapten that can be added to 5' amino linker oligos using the Boehringer DIG kit.
A variety of fluorescent labels attached to oligos are available and more are being added. These can be used in a variety of detection schemes and are particularly useful for automated fluorescent sequencing.