Sequence Troubleshooting
The following is taken from pages 3-12 and 3-13 of the DNA
Sequencing: Chemistry Guide Part Number 903563, Version A, May
1995. © Copyright 1995, The Perkin-Elmer Corporation.
Although base calling is easiest for the analysis software
when signal strength is high, good signal strength does not always go
hand-in-hand with high quality data. Background noise can obscure the
true sequence data.
Dirty DNA templates are the most common cause of noisy data.
Some template preparation procedures are not compatible with
fluorescent sequencing. A template that gives poor sequence is usually
said to be dirty if it contains some impurity that inhibits the
sequencing enzyme or that inactivates some other reagent in the
sequencing reaction. The contaminants are usually small molecular
weight species, proteins, or RNA. Templates that produce poor data can
sometimes be purified to a quality that yields good data. Please
contact us if you require information on template clean-up techniques.
If two or more different DNA templates are present in the
reaction (a mixed template) and all possess a primer annealing site,
sequence data from each will be superimposed in the electropherogram
giving a superimposed peak pattern.
It is a good idea to check each template preparation by
agarose gel electrophoresis to determine its purity. When purifying
recombinant plasmids in bacteria, plate out the transformants to obtain
isolated colonies. Then select a single colony and restreak out on a
plate to again select the colony for growth of cultures. If isolating
bacteriophage DNA by growth from plaques, pick from a fresh plate and
choose an isolate that is well away from others. If sequencing a PCR
fragment, check the product on a gel to ensure that only one product
was formed in the PCR or use a sequencing primer nested within those
used for the amplification.
Enzyme slippage can occur in homopolymer regions (such as the
poly(A) tails present in cDNA), thus skipping a base or incorporating
an additional one. The sequence beyond the homopolymer region may then
be shifted by one or more bases giving the appearance of multiple
overlapping sequences in the electropherogram or stuttering peaks.
Lastly, multiple priming events (either by n-1 primers, by a
different contaminating primer or a single primer annealing to more
than one site) can lead to multiple overlapping sequences.
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