Facts about oligonucleotides
Principles of oligo synthesis
and potential problems
Oligonucleotides are synthesized in vitro in the opposite direction
than in the process of in vivo synthesis, it means from the 3Žend to
the 5Žend. The synthesis is performed in a column carrying the first
nucleotide in the 3Žend of the synthesized sequence. In subsequent steps,
other nucleotides are bound behind the first nucleotide according to
the desired sequence defined by an operator of the synthesizer.
Incorporation of each nucleotide into the sequence of synthesized oligo
includes about 100 steps - the exact number depends on the scale of
the synthesis. A crucial reaction is coupling, in which one nucleotide
monomer is coupled to an oligomer, which is immobilized in column, resulting
in an oligomer that is one nucleotide longer.
Besides the coupling, three other chemical reactions are necessary to
prepare the growing chain of oligomer for the following coupling: detritylation,
capping, oxidation.
To ensure best yield of synthesis, it is very important for detritylation
and coupling to occur very quantitatively.
On the other hand, capping is crucial for the resulting quality of the
product. Detritylation prepares the oligomer for reaction with the next
monomer in the coupling. Coupling is a type of reaction that occurs
with slightly less than 100% efficiency. When a little part of the oligomer
chains does not react with the monomer, they are able to react in a
subsequent coupling resulting in a chain with one nucleotide missing.
Such an oligomer has a false sequence with one missing nucleotide, which
strongly decreases the quality of the synthesized oligonucleotide. Capping
is the reaction that terminates the chains that did not undergo coupling
and prevents their subsequent prolongation.
In other words, if a coupling does not work with a sufficient quantitative
rate, a synthesis yields oligonucleotide containing an unfavourable
ratio of perfect chains in relation to uncompleted shorter chains. If
a capping does not work quantitatively, in addition to perfect and uncompleted
chains, the resulting oligonucleotide will contain false chains with
inauthentic sequences with missing nucleotide/s in their sequences.
A quality oligonucleotide does not contain any inauthentic sequences
at all.
The quality synthesis depends on many factors and its efficiency is
around 99%, never higher under current conditions. Tab. 1 shows the
rate of perfectly synthesized chains of full-length oligonucleotide
depending on the length of the oligonucleotide. The conclusion is that
the oligonucleotide product of high quality always contains an important
rate of uncompleted chains: the longer the oligonucleotide, the higher
the rate.
If orders of lengthy oligonucleotides are planned it is important to
take into account that only a relatively low rate of full-length oligonucleotide
will be present in the resulting product. The simplest way, in which
to remove uncompleted chains from the product is to apply OPC purification.
However, it is not necessary for most methods.
In GENERI BIOTECH, the efficiency of coupling is monitored in the synthesis
of every oligonucleotide by measuring conductivity of detritylation
by-product.
After the synthesis, chains of oligonucleotide are cleaved from the
solid support in the column, most often by using concentrated ammonia
solution. In the same step, deprotection of nucleotides occurs, which
means that protective moieties are removed from the nucleotides that
prevented the oligonucleotide from undesired branching amino-moieties
during the synthesis. Deblocking of phosphate moieties also results
from deprotection. After the deprotection, a purification of the oligonucleotide
comes.
Estimated correlation
of the percentage rate of the full-length product on the efficiency of
synthesis and length of oligomer
| Efficiency of synthesis |
length of oligonucleotide (number
of nucleotides) |
| 20-mer |
30-mer |
40-mer |
60-mer |
| 99,5% |
91% |
86% |
82% |
74% |
| 99% |
82% |
74% |
67% |
55% |
| 98% |
67% |
55% |
45% |
30% |
| 97% |
54% |
40% |
30% |
16% |
Quality control of oligonucleotides
Control of UV spectrum (as a curve of absorbance depending on the wavelength
of passing UV light) of each oligonucleotide synthesized is a standard
type of control. This kind of control is able to reveal only the presence
of chemical impurities; it does not reveal any presence of chains with
the undesired lengths in the product that influences quality of the
oligonucleotide.
The most effective control of an oligonucleotide is HPLC control (trityl-off
HPLC). The resulting chromatogram gives evidence of the presence of
uncompleted chains and other by-products from a synthesis, including
imperfectly deprotected chains. If a synthesis of labelled or modified
oligonucleotide is performed, a chromatogram can even reveal the presence
of unlabeled or unmodified chains in the resulting product and enables
the quantification of the ratio of modified to unmodified oligonucleotide.
If an HPLC quality control of oligonucleotide is ordered, a record
of chromatographic analysis of the oligonucleotide with a description
of the peaks on it is included.
PAGE quality control of oligonucleotides is not provided any more because
of it being obsolete. It has been replaced by the much more effective
HPLC quality control.
Purification of oligonucleotides
When oligonucleotides are purified, two methodologically different problems
are solved:
-
removing chemical impurities (both, organic and inorganic)
-
removing uncompleted chains of shorter lengths.
There are a few basic methods of purification of oligonucleotides and
manufacturers use all of them. However, the quality of resulting products
is quite different.
Review of main purification methods:
Methods that d o n o t r e m o v e
uncompleted chains of shorter lengths
-
simple evaporation of ammonia solution of the
synthesized oligonucleotide with no subsequent step. In this case,
a user gets all the yield of oligo, but the product still has impurities
from the synthesis and it is in the form of ammonia salt that makes
it less soluble and therefore impropriate for some applications. This
method is less laborious and less expensive. GENERI BIOTECH does not
offer this method of purification.
-
purification by precipitation - it is similar
to the generally used precipitation of DNA. It results in a product
of good quality that does not contain many impurities. The oligonucleotide
is in the form of sodium salt and that is the most appropriate. When
this method is applied, the resulting yield of oligo can be decreased
from one half to two thirds of crude product in a substandard way.
GENERI BIOTECH does not offer this method of purification.
-
separation of oligonucleotide in Sephadex column,
this enables it to retain only oligonucleotide chains containing minimal
amounts of impurities. It results in a pure sodium salt of oligonucleotide;
however, quantity loss and lower yields are a drawback of this method.
A higher yield can be simply achieved, but at the cost of a decrease
in the purity of the resulting product. Oligonucleotides synthesized
in GENERI BIOTECH are currently purified on Sephadex G-25, DNA grade
(if either an OPC or HPLC purification is not ordered).
Methods that do r e m o v e uncompleted chains
of shorter lengths
Quality oligonucleotides of normal lengths do not contain
high rates of uncompleted chains. Such a rate influences substantially
the quality of the oligo (in addition to absence of salts and impurities
from cleavage and deprotection). A quality oligo of normal lengths does
not require additional purification when used in most molecular applications.
Yields of synthesis
It depends on the length of the oligo, the scale of synthesis and what
purification method was used. Generally, the longer the oligo and the
more efficient purification method was used, the lesser the yield. If
an oligo is consumed repeatedly in repeated techniques (e.g. in routinely
and frequently performed one type of PCR) we recommend that the synthesis
be performed at a scale of 200 nmol. Synthesis at a scale of 200 nmol
is about 50% more expensive than at a scale of 40 nmol, but its yield
will be at least three times higher.
The yield of the synthesis depends substantially upon the ratio of nucleotides
in a chain: the yield of oligos comprising of a high rate of G and C,
that generally have lower coupling, will be apparently lower than in
oligos containing high rate of A and T. The lowest yields are usually
observed in chains of pure polypurins.
Degenerated oligos
Oligos are synthesized with no additional charge when the degeneration
is achieved by a simple mix of nucleotides in designated position. However,
one exception exists: if degenerated position is located in the 3Žend
of oligo, the technique and price of a the synthesis will differ. In
this case, it is necessary to manufacture a special column with an anchored
mixture of nucleotides first. For this reason, when degeneration on
the 3Žend is ordered, it can be performed only at a scale of 200 nmol
or higher and an additional fee is charged. More detailed information
is stated in chapter Degenerated
oligonucleotides. Abbreviations
for degenerated oligos.
Extremely short or long oligonucleotides
Very short oligonucleotides: oligonucleotides 2 to 16 nucleotides
long are considered to be very short. The price is calculated in the
usual way (price per one nucleotide multiplied by number of nucleotides
in the oligo) and is extended by surplus
also taking into account the price of the synthesis column with anchored
first nucleotide. (For details, see chapter Pricelist
of oligonucleotide chains).
Extremely long oligonucleotides are ones longer than 50 nucleotides.
Their price calculated in the usual way (price per one nucleotide multiplied
by number of nucleotides in the oligo) is multiplied by a coefficient,
which reflects the different technology used in synthesis of long oligos,
including the higher probability that resynthesis needs to be performed.
Syntheses of oligos up to 99 nucleotides long are currently performed.
Longer oligo can be ordered only after consultation.
If a synthesis of an oligo longer than 40 nucleotides is required, we
recommend synthesis be performed at a scale of 200 nmol followed by
OPC purification: when not provided, the resulting product can contain
high rates of uncompleted chains that may interfere in some of the applications.
Oligonucleotides with potentially
restricted solubility
Oligonucleotides of some specific sequences are not possible to be resolved
after they are dried as they form either insoluble or hardly soluble
complexes. Therefore, we supply oligonucleotides of these suspect sequences
in a solution with a warning that this phenomenon could occur if they
have been dried.
Stability of oligonucleotides
Oligonucleotides are very stable compounds keeping their biological
activity for many years (e.g. capability of PCR amplification) if stored
in temperatures under -20°C. It is generally assumed that frequent melting
and freezing lead to their decreased function. For this reason (and
for the reason to try to limit probability of contamination by PCR amplicons),
it is recommended to store them in aliquots.
On the other hand, our own experience shows that oligos remain stable
even when they were subjected to very inappropriate treatment (see details).
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