H i g h e r Q u a l i t y ... B e t t e r R e s u l t s
Troubleshooting Guide
Mutations in
Oligonucleotide Sequences
www.microsynth.com
Higher Quality ... Better Results
Introduction
It is generally known that the occurrence of mutations during the synthesis of oligonucleotides can’t be entirely
avoided. Oligo producers can only minimize the risk factors and thus try to keep them at a low or negligible level.
One reason for mutations are specific regions within an oligonucleotide sequence that favor the formation of
secondary structures such as stem-loops (hairpins), tetraloops or pseudoknots. These secondary structures may
then preferentially impair specific chemical reactions during the chain assembly cycle at precisely such locations
and as a consequence increase the likelihood for a mutation. Another reason for mutations may arise from general
limitations in chemistry and synthesis technology. It is important to note that mistakes from human error can be
almost excluded since the production processes for oligonucleotides nowadays show a high degree of automation.
Highly automated synthesizer technologies and purification methods are operated in combination with
sophisticated, completely barcode-driven labour management software systems.
Mutations within oligonucleotides typically affect only a small portion of the molecule, are spread randomly over the
sequence and occur at very low levels. In other words, the vast majority of the synthesized full-length oligos
corresponds to the targeted sequence. Mutations occurring at a low background are usually not a problem for
applications like sequencing, hybridization, gel shift etc. However, such concealed mutations may become visible
when oligonucleotides are used for molecular cloning.
Since we know that mutations within oligonucleotides are especially frustrating during molecular cloning projects,
we have worked out this troubleshooting guide in order to support you to design your future experiments in the
most optimal way. Moreover, Microsynth’s application specialists are pleased to answer questions you may have
either about the information in this troubleshooting guide or about the use of oligonucleotides for molecular biology
applications in general. Please get in contact with us via: [email protected].
Observation
Possible Cause and Recommended Solution
5’ truncated oligonucleotide
PCR artifact, unpurified primers
sequences after cloning and
sequencing
Preferred priming from 5’ truncated oligonucleotides during PCR is an artifact
that we have observed many times. Basically, there are three possibilities to
minimize or deal with this artifact: 1) If for example, the first two clones did not
show the expected result, pick another couple of clones and sequence them.
You may have used standard primers which have been desalted only and
therefore exhibit a low level of purity. In such a situation it may be more
reasonable to sequence another 3-10 clones before repeating the time
consuming process of oligonucleotide re-synthesis, PCR and cloning. 2) Do not
perform your PCR overnight. Even if you keep the finished PCR reaction at 4°C,
the 5’ exonuclease activity of your polymerase may not be zero and lead to
degradation of your PCR amplicon. 3) Use HPLC or PAGE purified
oligonucleotides for your next cloning experiment. By investing a little bit more
money in advance you may save time and money at the end. By means of
2
Microsynth Troubleshooting Guide – Mutations in Oligonucleotide Sequences
Higher Quality ... Better Results
applying HPLC (<50mers) or PAGE purified oligonucleotides (>50mers) you
simply start with an oligonucleotide fraction where side products (n-x) have been
largely separated.
Restriction enzyme does not
PCR artifact, unpurified primers
cut my PCR product
Recommended solution is same as above. However, please be aware that
some restriction enzymes do not work efficiently close to the end of a DNA
fragment. In such cases, we suggest that you extend your primer sequence by 3
to 6 bases in front of the restriction site.
Mutations at non-specific
PCR artifact due to the use of a DNA polymerase exhibiting a relatively high
sites within oligonucleotide
error rate (e.g. Taq DNA polymerase)
sequences after cloning and
sequencing
For routine PCR, where simple detection of an amplification product or
estimation of the product's size is important, Taq DNA polymerase is the
obvious enzyme to choose. However, when the amplified product is to be
cloned, Pfu DNA Polymerase is a much better enzyme of choice for PCR. Pfu
DNA Polymerase exhibits one of the lowest error rates of any thermostable DNA
polymerases studied (~1 error in 1.3 million base pairs duplicated).
Mutations at specific sites
One or more hairpin structures within the oligonucleotide sequence
within oligo sequences after
cloning and sequencing
Hairpin structures increase the likelihood for introducing mutations within
oligonucleotides during their chemical synthesis. However, the likelihood of
inserting mutations at or close to secondary structures is much higher during the
PCR reaction due to steric hindrance of the polymerase. In such cases, the
percentage of correct clones will be significantly lower. In order to pick the right
one, we recommend you to sequence a higher number of clones. Another
possibility may be to (partially) disintegrate the hairpin by introducing silent
mutations (make use of the redundancy of the genetic code).
Multiple-base deletions
Long, unpurified primers (>50mers)
within oligo sequences after
Lethal protein  selection for mutations
cloning and sequencing
Generally, multiple-base deletions are stretches of 2-20 bases that are lacking
within an oligonucleotide sequence.
The longer an oligonucleotide sequence is the more difficult it is to synthesize it.
Long, unpurified oligonucleotides not only show a significant contamination with
n-x products. With increasing length of the oligonucleotide the likelihood for
single-base but also multiple-base deletions accumulates. In such cases we
3
Microsynth Troubleshooting Guide – Mutations in Oligonucleotide Sequences
Higher Quality ... Better Results
recommend you to order new primers that have been purified by means of
PAGE. At Microsynth, we have made the experience that oligonucleotide
mixtures containing multiple-base deletions will be completely eliminated
following PAGE purification.
Another reason may be that the recombinant protein produced within E.coli is
harmful or even lethal to its host and therefore favors the growth of clones
carrying mutants. Again, we recommend in the first instance to increase the
number of clones to be sequenced.
Some Examples
Below we show 3 representative examples about specific problems during cloning projects related to mutations
within oligonucleotide sequences.
Example 1: 5' truncated Oligonucleotide Sequences after Cloning and Sequencing
Customer: “I have done PCR with two desalted 30mer primers from your company. Following cloning and
checking one clone by means of sequencing we observed that the resulting sequence did not contain the first 6
bases of the forward primer at the 5’end. In contrast, the reverse primer was completely correct.”
Microsynth’s research: A long oligonucleotide was synthesized as a template for PCR matching the above
mentioned 30mer forward and reverse primers. The 30mer forward primer was synthesized again and further
purified by PAGE. Thereafter, PCR was performed with both primer groups using the high-fidelity polymerase Pfu.
Finally, the blunt-end PCR products were cloned, 29 respectively 35 clones were sequenced and the missing
bases of the forward primer were counted. The result of this experiment is shown in the following table:
Number of
missing bases
at the 5' end
0
1
2
3
4
5
6
7
8
9
10
Total
Complained oligo (desalted only)
New oligo (PAGE-purified)
Number of clones
%
Number of clones
%
16
6
3
2
0
0
1
4
1
1
1
35
46
17
9
6
0
0
3
11
3
3
3
100
23
4
0
2
0
0
0
0
0
0
0
29
79
14
0
7
0
0
0
0
0
0
0
100
Obviously, PAGE purification of the forward primer clearly improved the number of correct clones (46%  79%).
This tremendous effect related to PAGE purification was unforeseeable when comparing the MALDI-TOF spectra
of the desalted and PAGE purified oligo (no significant n-6 peak was visible; data not shown). Thus the customer
4
Microsynth Troubleshooting Guide – Mutations in Oligonucleotide Sequences
Higher Quality ... Better Results
unfortunately picked one of the rare n-6 truncated clones. Instead of picking only one clone he should have
sequenced at least 3-5 clones in order to find the correct one already in the first instance.
Example 2: Mutations in Hairpin Oligonucleotide Sequences
Customer: “We have cloned long (50-80mer) oligonucleotides and sequenced 3 clones. All of them showed a
mutation at the same position. The oligonucleotide shows a strong hairpin and is used for siRNA technique.”
Microsynth’s research: Following 79mer oligonucleotide containing a 7-base hairpin (highlighted in grey) was
synthesized on two different oligosynthesizers operating with different fluidic systems. Whereas Oligo No. 1 was
produced at Microsynth (Producer 1), Oligo No. 2 was synthesized externally (Producer 2).
5’CAAGAAGCGATGTCCTTGTCATCGCTAGAGCTATCTCCTAGCTGGATCGATCGATAAGGGATCTAGCCGATCTTGAGAT3’
Thereafter, above mentioned 79mer oligonucleotide was synthesized (using the same synthesizer as for
oligonucleotide No. 1) but with 3 bases (bold & underlined) exchanged. The aim was to disrupt the hairpin
structure.
5’CAAGAAGCGATGTCCTTGTCAGTGTTAGAGCTATCTCCTAGCTGGATCGATCGATAAGGGATCTGGCCGATCTTGAGAA3’
At Microsynth, all three oligonucleotides were purified by PAGE and then PCR amplified using a high-fidelity
polymerase. After cloning, several (53 – 81) clones were sequenced and analyzed using an alignment program.
Mutation site
1 - 21
22
23
24
25
26 - 30
31 - 35
36 - 40
41 - 45
46 - 50
51 - 55
56 - 79
Total number of mutations
Number of sequenced clones
% of correct clones
Oligo No. 1 (hairpin)
Number and type* of
mutations
0
1 x Del, 1 x Sub
4 x Del, 12 x Ins
12 x Ins, 1 x Sub
2 x Del, 2 x Sub
1 x Del, 5 x Ins
2 x Del
0
0
1 x Del
1 x Del
1 x Del, 1 x Sub
47
53
17%
Oligo No. 2 (hairpin)
Number and type* of
mutations
0
0
0
42 x Sub
0
0
4 x Sub
1 x Del
5 x Sub
2 x Sub
5 x Sub
3 x Sub
62
63
14%
Oligo No. 3 (no hairpin)
Number and type* of
mutations
0
0
0
0
0
0
0
0
1 x Sub
3 x Del
0
1 x Del
5
81
85%
*Ins = Insertion, Sub = Substitution, Del = Deletion
This example clearly shows that oligonucleotides containing hairpin structures are susceptible to mutations. The
investigated oligonucleotides No. 1 and No. 2 have most failures in the positions 23 and 24 and almost all
problems could be eliminated with the oligonucleotide No. 3 which does not contain a 7-base hairpin (see also
images on page 6). The percentage of correct clones increased from 17% or 14%, respectively, to 85%!
Furthermore, another observation could be made from this investigation. The final outcome, i.e. the number of
correct clones, was essentially the same for both hairpin containing oligos No. 1 and 2, independently of the
5
Microsynth Troubleshooting Guide – Mutations in Oligonucleotide Sequences
Higher Quality ... Better Results
synthesizer technology used. Hence, the primary goal of any cloning experiment must be to recognize the
presence of hairpins within an oligonucleotide sequence in advance and to deal with any such risk (dissolve
hairpin and/or pick a higher number of clones for sequencing).
6
Microsynth Troubleshooting Guide – Mutations in Oligonucleotide Sequences
Higher Quality ... Better Results
Example 3: Effect of PAGE Purification on Multiple Deletions
General observation: Based on our own work, we observed that long, desalted oligonucleotides not only show
single-base deletions but multiple-base deletions as well. Multiple base deletions are stretches of 2-20 bases
which lack within an oligonucleotide sequence. In order to better understand the impact of different purification
methods we have performed the following experiment.
Microsynth’s research: Three different oligonucleotide pairs (No.1 - No. 3) with each consisting of a 81mer and a
79mer were synthesized. Both of them contained a 15 base stretch of complementary sequence at the 3’ end.
Following synthesis, a fraction of the total amount of oligonucleotide pair No. 1 and 2 was subjected to HPLC
purification whereas another fraction thereof was additionally purified by PAGE. Oligonucleotide pair No. 3 was
desalted only, and a fraction thereof was further purified by PAGE. Thereafter, the 6 different fractions of
oligonucleotide pairs No. 1 to No. 3 were annealed and PCR amplified with a high-fidelity polymerase. Following
cloning, several clones were sequenced and the mutations in this 145bp fragment were counted (see table
below).
Source of oligonucleotides / purity
Oligonucleotide pair No. 1 - HPLC-purified
Same as No. 1, but additional purification by PAGE
Oligonucleotide pair No. 2 - HPLC-purified
Same as No. 2, but additional purification by PAGE
Oligonucleotide pair No. 3 - desalted only
Same as No. 3, but further purification by PAGE
Number of
sequenced clones
58
68
66
83
81
68
Number of clones with multipledeletions (2 – 54 bases)
7
0
5
1
3
0
It can be concluded that PAGE purification has by far the largest effect on the final outcome of cloning
experiments. It can be further concluded that for long oligonucleotides HPLC purification does not have an
advantage over oligonucleotides that have been desalted only. This is in agreement with our general experience
that oligonucleotides should be subjected to HPLC purification only up to a sequence length of 50 bases.
Conclusion
In general, it can be summarized that many different parameters can influence the occurrence of mutations within
oligonucleotide sequences. This is especially true, if oligonucleotides are utilized for cloning experiments where
artifacts mainly during PCR and cloning contribute to the observed mismatches. In this context, it is important to
note that even under optimized experimental conditions, it is not realistic to expect a percentage of correct clones
higher than 90%. Therefore, we strongly recommend to first get an idea about the expected percentage of correct
clones when performing your experiment. Thereafter, you choose the number of clones to be checked for the right
sequence.
7
Microsynth Troubleshooting Guide – Mutations in Oligonucleotide Sequences
Higher Quality ... Better Results
Critical Information related to Oligonucleotides Purity Levels and Applications
The synthesis of oligonucleotides is carried out on a
solid support (controlled pore glass) in a series of
100.0
of the oligonucleotide) with each cycle comprising a
80.0
series
60.0
150
140
130
120
110
incomplete activation or coupling, respectively. The
0.0
90
reaction is the formation of n-x products due to
0.985
20.0
100
Using phosphoramidite chemistry, the main side
0.990
40.0
80
chemical reaction never processes completely.
0.995
70
A
60
oxidation).
50
(5’-deprotection,
40
and
30
reactions
capping
20
chemical
10
of
activation/coupling,
Yield [%]
cycles (number of cycles corresponds to the length
Oligonucleotide Length
longer the sequence, the more n-x products occur.
The figure on the right hand side gives an overview how the degree of the coupling efficiency (CE) and the length
of the oligonucleotide (n) influence the final yield of the raw product (Yield = CEn-1). Thus, the theoretical yield of a
full length product for a 50mer at 99.0% coupling efficiency will only be 61%. In general, the purity level that you
require depends on the effect that alternative sequences (n-1, n-2, … n-x products) will have on your specific
experiment. In the following a short summary is given about the various purification levels that Microsynth offers
you as a customer. Furthermore, we specify the required purity level for applications involving unmodified oligos.
Desalted Oligonucleotides
All our oligos are at least desalted to largely remove residual low molecular by-products arising and accumulating
from the frequent chemical reactions during synthesis. Such purification is sufficient for oligonucleotides shorter
than 30 and/or oligonucleotides used for non critical applications such as: PCR, sequencing, probing, mobility
shift or hybridization. However, desalted oligos are not recommended for use in molecular cloning projects.
HPLC Purified Oligonucleotides
Oligos <50 bases in length can be well purified via Reverse Phase HPLC. Through this purification approach,
preferably residual, n-x truncated oligos (lacking the hydrophobic DMT protection group at the 5’ end) are
removed. This results in a 90-95% purity of the targeted oligonucleotide. RP-HPLC is useful for a higher level of
purity required for more demanding applications such as: cloning, DNA fingerprinting, Real Time PCR, FISH etc.
PAGE Purified Oligonucleotides
Polyacrylamide gel electrophoresis (PAGE) purification is generally necessary for long oligos (>50 bases) and for
all those primers with critical 5' sequences (restriction endonuclease sites, RNA promoters). It is the best method
to differentiate full-length oligos from aborted sequences (n-1 oligos), based on size, conformation and charge.
PAGE purification has an excellent resolution and yields a product that is, on average, 95-99% pure. This type of
purification is highly recommended for sensitive experiments such as: cloning, mutagenesis, DNA fingerprinting,
in situ hybridization, gene synthesis etc.
Quality Control
Above all, a stringent quality control system ensures the consistently high quality of all our oligonucleotides.
Microsynth performs online trityl monitoring of all oligonucleotides in order to control the coupling efficiency after
each cycle. Following synthesis, molecular identity of our oligonucleotides is either checked by MALDI-TOF (up to
50 bases) or by analytical PAGE (51 to 125 bases).
8
Microsynth Troubleshooting Guide – Mutations in Oligonucleotide Sequences