Section II: Troubleshooting a CEQ System Problem
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CEQ™ 8000 Series Genetic Analysis System
Insertions Caused by Mixed Templates,
PCR Primer Carryover or Primer Mis-priming
In many instances poor sequencing results are the cause of multiple sequencing events
occurring in one sequencing reaction. These multiple sequencing events are usually
caused by either mixed templates (2 or more unique templates in a reaction), PCR
primer carryover (sequencing reactions primed by the PCR primer in addition to the
sequencing primer) or mis-priming of the sequencing primer (the sequencing primer
binding to 2 or more sites on the template DNA). All three types of multiple
sequencing events have similar symptoms: inserted bases, more than 8–10 bases per
minute in the raw data and “peaks under peaks” in the raw and analyzed data (more
than one dye labeled sequencing fragment co-migrating).
1.
When two or more different populations of DNA molecules with a common
priming site are present in the template DNA preparation multiple sequencing
events will occur. It is very common to obtain a mixed template from PCR
samples cut from gels (e.g. differential display) or from PCR amplified template
that contain more than one amplified fragment. It is also seen with plasmid
samples when, for example more than one recombinant colony is inoculated into
a single culture. In this case, the initial analyzed data is good (common vector
sequence) but the inserted region is different and will generate inserted bases.
2.
PCR primer carryover and sequencing primer mis-priming are similar in that
they will generate inserted bases from multiple primer extension events
occurring in the same sequencing reaction. In the former case not all of the
primers used to generate a PCR product were removed prior to doing the
sequencing reaction and so these leftover primers now initiate a sequencing
reaction from places on the template that are different from that of the desired
sequencing reaction.
3.
In the case of sequencing primer mis-priming, there are 2 or more binding sites
on the template DNA for the primer to initiate the sequencing reaction. Primer
mis-priming results in too many bases being present in the analyzed data, which
then generates inserted bases in the sequence. In some instances the data has so
many insertions that the sequence analysis will fail.
