Troubleshooting Poor or Failed
Sequencing Results
Your samples may fail to produce
any sequence data. Also, data of
some samples that you do receive
may be of poor quality. This may
be due to the following reasons.
Good quality data
Sharp peaks characterize DNA
sequence of high quality with
little to no background. A good
quality data looks like this.
Incorrect Primer or Template concentration
This is one of the main reasons
samples fail to generate little
or no data. You may want to try
a richer growth media such as
Terrific Broth or a more robust
mini-prep. The primer
concentration should be as
mentioned in the request form.
Both high and low concentrations
of the DNA may cause problems in
generating sequence data.
The following is a chromatogram
of a sequence where the
concentration of the sample is
too high.
Poor quality DNA template or Poor purification
Next to insufficient template
concentration, poor quality DNA
is the primary cause of poor or
no results. The AmpliTaq
polymerase used for fluorescent
sequencing is much more
sensitive than standard Taq and
hence requires much cleaner
template DNA. The following
sequence is dirty and many peaks
are not distinguishable from the
background.
Primer binding to multiple sites
If your results appear to have
multiple peaks per base pair,
there may have been multiple
binding. You may need to
re-design your primer and
utilize commercially available
software to design primers. It
is vital to ensure that the
primer sequence will not
self-hybridize. Also, primers
that fail to bind to the
template due to an insufficient
concentration will not yield
sequence data.
The following is an example of a
sample of a template containing
multiple priming sites.
N-1 Primer molecules
The presence of small peak
under a large peak in such a
way that it resembles the
stutter of the following peak
shows the presence of N-1
primer in your primer
solution. If you were able to
separate the two peak data you
will see the two same
sequences with only first base
short in the small peak data.
Contaminants Within Your Template DNA
The presence of EDTA in your
template DNA will inhibit the
cycle sequencing reaction, as
will the presence of high salt
concentrations. Capillary
electrophoresis is much less
forgiving of salt present in DNA
as compared to other sequencing
methods. Cellular debris, RNA,
and phenol also inhibit this
reaction. Please be certain to
submit your samples in water,
not TE, and refrain from
protocols calling for phenol:
chloroform extractions.
The following chromatogram shows
a sequencing sample with a
moderate amount of contaminants,
which can be seen running along
the base of the peaks.
Prime Melting Temperature Is
Incorrect
All primers should have a
melting temperature between
50-65 °C. Several methods and
all commercially available
primer designing programs can
determine the correct Tm.
Template Does Not Contain a
Sequence Complementary to the
Primer or the Primer Does Not
Work with Flourescent
Sequencing
Occasionally one of the primers
used to generate the PCR product
does not work in fluorescent
cycle sequencing. It is likely
that such a primer, although
sufficiently competent in the
exponential PCR process, is very
inefficient in the linear
amplification of cycle
sequencing.
Sometimes a loss of signal will
occur after a poly nucleotide
region (especially G's or T's).
There are two options. First,
you could use a primer that sets
on the poly N region. If you get
through and know the final base,
a primer can be designed with 17
like bases coupled with the
known base, or make it a mixed
base site if it is unknown (GGGGGGGGGGGGGGGGGN).
The second option is to try
sequencing from the reverse
direction.
Below are two samples that have
a repetitive poly T region with
poor sequence that follows it.
