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TROUBLESHOOTING
The Facility uses strict protocols and internal quality controls
to insure our reactions are as consistent as possible. Most often when
there is trouble with the sequencing reaction, the cause lies with the
DNA template itself, making it very important that the materials we
sequence are of the highest quality possible.
Multiple
Overlying
Problem:
Sequence appears as multiple overlying traces, not always throughout the
entire sequence How to Identify : Sequence data
begins to show multiple overlapping traces after a point in the sequence,
although the sequencing signal remains strong. A compression phenomenon
and contaminated DNA prep can look similar, though the contaminated prep
will typically show overlapping traces immediately after the insert
cloning site.
Cause:
DNA fragments of different size with the same electrophoretic mobility,
ie fragments that migrate on top of each other during electrophoresis.
This phenomenon is thought to be caused by regions of secondary structure
within the template DNA, which are often, but not always, found in
regions with a high G/C or high A/T content.
Solution:
1. Repeat sample using DMSO or Glycerol
2. Incubate the reaction at 96C for 10 minutes before
cycle sequencing.
3. If the sample is a PCR product, try amplifying the
DNA with substitution of 7-deaza-dGTP for 75% of the dGTP in the PCR, and
then sequence the PCR product.
4. Choose a new primer close to the compression site
which can help avoid the effects of the secondary structure.
Contaminated
Plasmid DNA Preparation
Problem:
Contaminated plasmid DNA preparation leads to overlapping peaks in the
sequence data.
How To
Identify: Sequence data is clear out to the end of the
insert cloning site, at which point peaks underneath other peaks can
often be seen. In the worst case, the peaks will obscure each other,
making the entire sequence unreadable.
Cause: Picking
more than one clone for a single plasmid preparation, or picking a single
colony that contains two or more plasmids, each with different inserts.
Solution:
1. Carefully pick a single colony from your plate into
your growth medium in a clean environment.
2. Verify that your single colony contains your insert
of interest after DNA preparation by restriction digest and agarose gel
analysis.
Gradual Early Termination of
Sequencing Signal
Problem:
Premature termination of sequence due to repetitive structure(s) of DNA.
How to identify : Repetitive
stretches of DNA sequence (usually mono- or dinucleotide repeats) that
show strong sequencing signals at the beginning of the repeat and then
gradually taper off to an unreadable signal.
Cause:
Various ideas have been put forth on this subject: (1) Slippage of the
DNA polymerase on the template strand during elongation (2) Formation of
secondary structure due to the repeat. (3) Depletion of dNTPs.
Solution:
1. Sequence the template from the other side of the
repeat in the reverse direction to obtain sequence on both sides of the
repeat. Be aware that it will be difficult to determine the precise length
of the repeat if neither sequencing reaction manages to span the entire
length of the repeat. Although it may not often be practical, using
different sequencing chemistries may improve results as well.
N-1, N-2, N-3... Priming Sequencing
Trace
Problem:
Sequence shows 'double peaks' throughout, with the second (generally
smaller) peak being the same base as that of the true base immediately to
the right of it.
How to
identify: Although an n-1 primer problem may look like
compression or priming of two different regions of DNA template, it can
be readily identified by each position containing the true peak and the
peak immediately to the right of it.
Cause:
1. Poor purification of the oligonucleotide during
primer synthesis which leaves a certain percentage of n-1mers in the
final product. When the DNA template is sequenced, this percentage of
n-1mers will prime the DNA template, causing some of the sequence to be 1
bp shorter than it should be. Primers that have begun to degrade will
also do so from the 3' end, causing a proportion of the original
sequencing primer to become n-1.
2.The primer has started to
degrade from the 3' end so that a portion of the sequences will have a
sequencing ladder that is one basepair longer than a non-degraded primer.
Solution:
1. Poor primer binding sight on the template. Solution : Cartridge purification of
oligonucleotides and even HPLC purification will help insure that n-1
priming of sequence doesn't occur. In both cases, re-synthesis of the
oligonucleotide is necessary.
No Readable Sequence Data
Problem:
Lack of sequence data
How to
identify: Sequencing trace is non-uniform. Unincorporated
Dye Terminator
Peaks at the beginning of the
sequence are out-of-scale. Basecalls contain many ambiguous bases ('N').
Cause:
1. Primer not annealing to template (no priming site).
2. No DNA template present in sequencing reaction.
3. DNA template is of poor quality.
Solution:
1. For (1), design a new primer which is free of
hairpins, free of duplexes, and has a Tm greater than 55C.
Secondary Structure
 Problem: Sharp drop or premature termination of sequencing signal
during the sequencing run.
How to
identify: Sequencing signal drops off sharply or terminates
before the end of the sequencing run (termination of sequencing signal
also occurs with PCR products, depending on how long the PCR fragment
is).
Cause:
Secondary structure in the template can cause various anomalies that
result in inefficient chain elongation after the region of secondary
structure.
Solution:
1. Many of the same techniques used to eliminate
compression can be helpful in obtaining good sequence data past regions
of secondary structure. In this case repeat
this sample adding 5% glyserol or DMSO.
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