Product Description
OLIGONUCLEOTIDES
Oligo
A short piece of
synthetic DNA is called an oligonucleotide or just an oligo. Oligos are
known as primers or probes, referring to their use in PCR or hybridisation
experiments, respectively.
Synthesis
The synthesis of oligos is performed on a DNA synthesiser, mostly on commercial instruments, but also more and more on own designed high throughput synthesisers. At TAG Copenhagen different types
of synthesisers, all dedicated to the job they shall do: Synthesising of
short oligos, long oligos, bigger amounts of oligos, oligos with
modifications etc.
Chemistry
Oligos are made in small columns filled with a solid support, usually glass or polystyrene beads, which are designed to anchor the growing DNA chain. The DNA synthesis consists of a series of chemical reactions:
- Deblocking: The first base from 3'end is via a chemical linker attached to the solid support. The trityl protecting group on 5čOH is removed with an acid to produce a free 5' OH ready for reaction with the next base.
- Coupling: The next base is added and coupled to the first base.
- Capping: 1-2% of the free 5'OH is not reacted with the next base and is then capped with acetic anhydride. These failed bases will play no further part in the synthesis cycle.
- Oxidation: The established phosphite group between the 1. and the 2. base is oxidised with iodine to phosphate.
- Deblocking: The trityl group on 5'OH on the 2. base is removed to produce a free 5' OH ready for reaction with the next base.
Each cycle of reactions results in the addition of a
single base. In this way a chain of bases can be connected by repeating
the synthesis cycles till the desired length is achieved. A computer
controls the reagent delivery system.
Coupling efficiency
The yield
of each step is called the coupling efficiency. No chemical reaction is
100%, but by using chemicals of high quality and by constantly trimming
the instruments, the coupling efficiency can be as high as 99%.
Trityl group
Every single DNA
base added in the DNA synthesis has a dimethoxytrityl (trityl) protection
group attached in order to to protect the 5'OH during the cycle of
chemical reactions. The trityl group is only removed immediately before
the next base is added.
Measuring of coupling
efficiency
When the trityl group is removed with acid (ex.
trichloroacetic acid in methylene chloride) a strong colour is generated.
The intensity of this colour is measured by UV spectrophotometry. From
this measurement the coupling efficiency is calculated and shows the
quality of the synthesis.
The table shows the effect of a difference in coupling efficiency when an oligo is synthesised.
|
% yield of a
n-mer |
99.5%
Coupling |
99%
Coupling |
98%
Coupling |
|
20 bases |
90 |
82 |
67 |
|
40 bases |
82 |
67 |
45 |
|
60 bases |
74 |
55 |
30 |
|
80 bases |
67 |
45 |
20 |
|
100 bases |
61 |
37 |
14 |
Purification
Standard deprotected oligos
Only a few
oligo houses continues to offer oligos Standard deprotected, which mean
that the synthesised oligo is treated with aqueous ammonia in order to
deprotect all amino functions in the chain, quantified and evaporated to
dryness. Often are these oligos suitable in many PCR reactions and
sequencing applications without further purification.
Desalted oligos
Oligonucleotides are desalted using ethanol in the presence of sodium
acetate. Desalting is acceptable for unmodified PCR primers but is not recommended for
modified or long oligonucleotides (>50 bases) because it cannot effectively remove
truncated failure sequences.
Cartridge purification - Reverse Phase Column
Cartridge purification is a reverse phase chromatography based purification method which removed salts and failure sequences from the synthesised oligo. The oligo is synthesised with the final trityl group on and only the full length oligo has the trityl group. Thus the full length oligo is retained in the cartridge, while failure sequences flow through. The trityl group is then removed allowing the full length product to be eluated. Some oligo houses calls this purification HPLC. At TAG Copenhagen this is the minimum
purification.
RP-HPLC purification
Reverse Phase High Performance Liquid Chromatography (RP-HPLC) is used to remove failure sequences or unincorporated label in the same way as a cartridge purification. Full length product with a Trityl group attached is retained in the HPLC column while failure sequences or unlabelled primers flow through. Full length product is eluated by slowly increasing the solvent concentration.
Please note that not all HPLC purified oligos from different oligo houses are
made according to the above mentioned procedure. It is possible to pass
oligo solution through an HPLC column very quickly using a steep solvent
gradient or in some cases no solvent gradient at all resulting in a lower
purity product.
At TAG Copenhagen the HPLC purification using a gradient is included in the price for all oligos longer than 50 bases and also included in the price for most of our 5'-modifications.
Page
purification
Polyacrylamide Gel Electrophoresis (PAGE) is a
method used to separate full length product from failure sequences based
on size and co-formation of the oligo.
Your choice
It is obvious that
the different purification methods gives you the option to choose the most
economical method for your experiment. In the below table you will find
the guidelines to suggested primer purity and explanations.
|
Application
|
Recommended purification |
|
PCR |
Desalted primers will normally work fine for standard PCR applications. If however non-specific amplification products are observed it may be necessary to try a higher purity primer since shorter failure sequences (n-1, n-2 etc.) may be competing with full length product.
|
|
Cycle Sequencing |
Desalted primers will work fine
|
|
Fluorescent Sequencing |
TAG Copenhagen recommends HPLC purification which will prevent unlabelled primers competing with fluorescent primers. TAG Copenhagen includes HPLC purification free of charge on all fluorescent primers.
|
|
Cloning experiments |
Full length primers will work best for efficient cloning. TAG Copenhagen recommends HPLC purification.
|
|
Mutagenesis |
HPLC purification is recommended by TAG
Copenhagen. Purified primers will result in the highest percentage
of successfully mutated clones, especially if the intended mutation
is at the 5'-end of the primer. |
OD reading
Optical density
reading at 260 nm (OD260) in an UV spectrophotometer is used to quantify
an oligo in solution. 1 OD is defined to be approximately 33 ”g single
stranded DNA. All DNA in solution included eventually failure sequences
are included in this reading.
Lower OD yields for purified
oligos
By removing failure sequences during a purification
procedure the OD reading decreases. A previous Table shows that for a 50
mer synthesized with 99% coupling efficiency approx. 40% of the DNA in
solution is sequences. When the failure sequences are removed through
purification the OD260 value measured would be at least 40% lower than for
the un-purified solution. Furthermore when performing HPLC or PAGE
purification some product is missed in order to ensure a pure final
product.
Scale of synthesis
TAG Copenhagen offers all international recognized scales of synthesis like 10 nmol, 40 nmol, 200 nmol, 1 ”mol scale etc. When ordering a 40 nmol scale approximately 40 nmols of the first base on the CPG is added to the synthesiser. For an average 25mer at least 25% of this starting material will result in failure sequences (see table). In addition to this, further losses occur during processing, transfer of material and quality control.
Variation of yields in delivery of oligos
Yields achieved from any given scale of synthesis is
depending on many factors as: Sequence, oligo length, instrument
efficiency, coupling efficiency, the amount of CPG added (this is a
manually step) and the wanted working-up procedure. In this way , no 2
syntheses are exactly the same. TAG Copenhagen guarantees a minimum yield
and will ensure that you receive at least this minimum yield.
Next day delivery of oligos - and
delays
Some time you order 6 or 8 oligos in one order and gets
with the next shipment only 5 or 7, respectively. At TAG Copenhagen we try
to ship oligos as soon as they are finished even if your order is not
complete. Often you will receive your oligos faster than we promise but
sometimes one oligo is missing. This missing oligo will be shipped as soon
as it is ready.
DNA synthesis is a complicated process and we are constantly improving the production, but still we have few losses, which is not approved by our quality control. Then we re-synthesise the oligo and you will receive the oligo a day later.
Recommanded maximum length of
oligos
When you look at table of coupling efficiency you will see
that with a coupling efficiency at 99% a 100-mer oligo would contain 37%
full length product and 63% failure sequences. A problem with small amount
of depuriation of base A during the synthesis gives even a lower yield of
full length. We recommend therefore customers to be modest in their wishes
and only order up to 100-mers, but of course we can make them longer. Please contact us.
Stability of oligos
As a guideline following table can be used:
Storage
State Temperature Maximum Storage Time
In Solution -20° > 6 month
In Solution Room Temp. < 1 week
Lyophilised -20° > 1 year
Lyophilised Room Temp. 1-2 month
Storage in solution should have a concentration >10 ”mol.
Oligos stored at room temperature or at 4° must be stabilised in TE and not only in water in order to prevent hydrolysis.
Repeated freeze/thaw should be avoided. A good idea is to aliquot the sample before freezing.
General Advise
When you order an oligo from different oligo houses you can be sure that the oligo has been made with the same chemistry with its limitations. Therefore:
- Order HPLC purified oligos for cloning experiments.
- When using longer oligos >50 bases for cloning purposes it may be necessary to screen an increased number of clones in order to find one with the correct sequence.
- Always sequence more than 1 clone and sequence across the primer before proceeding to downstream applications.
