Sanger Sequencing Troubleshooting Guide

Sanger Sequencing Troubleshooting Guide
Sanger Sequencing
Troubleshooting Guide
Below are examples of the main problems experienced in ABI Sanger sequencing. Possible
causes for failure and their solutions are listed below each example. The list is not
exhaustive so please contact us at [email protected] if you have any other
solutions to add.
Failed sequence
Problem
Lack of sequence data
Probable cause
No priming site present
Primers have degraded through
freeze-thaw cycles
Inefficient primer binding
Insufficient amount of DNA
template
DNA has degraded
Inhibitory contaminant in your
samples eg salts, phenol,
EDTA, ethanol
Solution
Make sure the primer site is
present in the vector you are
using
Redesign/ use a different
primer
Make up new primer stocks
Redesign primer
Quantify DNA
Increase the amount of DNA
template
Re-extract DNA
Clean-up DNA template
Sanger_troubleshooting_guide_v1.doc – August 2009
Weak sequence
Problem
Low peaks throughout
Probable cause
Insufficient amount of DNA
template
Inhibitory contaminant in your
samples (e.g. salts, phenol,
EDTA, ethanol)
Insufficient amount of primer
Inefficient primer binding
Solution
Quantitate the DNA
Increase the amount of DNA
template
Clean-up DNA template
Check primer dilution
Redesign primer
Poor start followed by weak sequence
Problem
Poor sequence at the
start followed by weak
signal
Probable cause
Primer binding to itself
Solution
Redesign sequencing primer
Other primers present
Check PCR clean-up has
removed all other possible
primers
Sanger_troubleshooting_guide_v1.doc – August 2009
Multiple peaks
Problem
Overlapping peaks in
the sequence data
Probable cause
Multiple priming sites
Solution
Use a different primer.
Residual primers (PCR product
has not been cleaned up)
Make sure all PCR primers and
dNTPs have been removed
Poor purification during
primer synthesis (full-length
primer is mixed in with
shorter primer missing one
base giving a shadow
sequence one base behind the
real sequence)
Order new sequencing primer,
preferably HPLC purified
Mixed plasmid prep
Contaminated template. Clean
sequence at the start with
mixed peaks beginning at the
cloning site
Ensure single colonies are
picked
Sequence the complementary
stand
Sequence from cloned PCR
products
INDEL in PCR product
Truncated sequence
Too much template
Sanger_troubleshooting_guide_v1.doc – August 2009
Secondary Structure
Problem
Sequence starts well
but signal stops
abruptly
Sequence starts well
but signal weakens
gradually (ski-slope
effect)
Sequence starts well
but signal weakens
rapidly
Probable cause
Secondary structure (GC and
AT rich templates can cause
the DNA to loop and form
hairpins)
Linearized DNA (restriction
enzymes may have cut an
internal site)
Too much DNA template
(overload of DNA leads to
excessive number of short
fragments)
Repetitive region (Repeat
regions, especially GC and GT
repeats, can cause the signal
to fade either due to
depletion or slippage or
secondary structure)
Solution
Add (1ul) DMSO to the
sequencing reaction to help
relax the structure.
Design primers close to the
hairpin
Run product out on an agarose
gel to check
Use less DNA template
Add (1ul) DMSO to the
sequencing reaction.
Sequence the complementary
strand
Multiple peaks downstream to homopolymer
Problem
Overlapping peaks
following stretch of
mononucleotide
sequence
Probable cause
Enzyme slippage occurs giving
varying lengths of the same
sequence after this region (n1, n-2 and n-3 populations)
Solution
Sequence the complementary
strand
Sanger_troubleshooting_guide_v1.doc – August 2009
Artifacts
Problem
Large peaks obscuring
the real sequence
Probable cause
Dye blobs caused by
unincorporated BigDye and
typically seen at 70bp and
120bp. Usually seen in failed or
weak sequences. Real sequence
can still be read underneath
these blobs
Solution
Add more DNA template or
less BigDye to sequencing
reaction
Sudden large
multicoloured peak
covering 1-2 bases
Sample peaks become
lumpy and increasingly
unreadable early in the
sequence (before
500bp)
Small air bubble of dried
polymer within the capillary
Contact us and sample can be
re-run
If related to individual samples
this is due to a contaminant in
the sample
Clean up template DNA
Degradation of polymer or
capillary array
Inform us if loss of resolution
continues
Sanger_troubleshooting_guide_v1.doc – August 2009
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