|
DNA Sequencing - Technical Corner
Troubleshooting
In our experience, the two most common reasons for sequencing failures
are:
1/ Presence of contaminant DNA (most often high molecular
weight chromosomal DNA) that was carried over during purification.
Typically, these contaminated samples will give rise to high background,
e.g., multiple fluorescent signal at any one base position. The
presence of contaminant may initiate non-specific priming in cycle
DNA sequencing reactions leading to a set of unrelated labeled DNA
fragments that will co-migrate with the bands of interest during
electrophoretic separation. This phenomenon may be observed with
unpurified primers as well. |
 |
2/ Presence of organic solvents (phenol, ethanol) or high
salt content. Typically, these samples will somewhat inhibit polymerase
reaction yielding lower fluorescent signals. In addition, the presence
of organic solvents and high salts will interfer with electrophoretic
separtion by distorting gel migration. The resulting signals are usually
impossible to interpret.
Please note that the use of degenerate primers (ie. Equimolar mixture
of same-length primers but differing by one to several bases) will result
in high background signals very much like in the case of contaminated
DNA (i).
The above two pitfalls can easily be avoided by:
- Using column-based miniprep kits.
- Carefully following miniprep protocols, especially for large vectors
where carry-over of high MW bacterial DNA is often observed.
- Verifying integrity and purity of your final product by standard agarose
gel electrophoresis.
- Washing your precipitated DNA pellet with 70% ethanol to remove co-precipitant
salts followed by a thorough evaporation of residual ethanol.
Accuracy Issue
The level of data accuracy in a DNA sequencing project is multifactorial
in nature including:
(i) Basic biochemical factors (ie. reaction mix, chemicals, enzyme processivity,
electrophoresis conditions) -Fig. 2
(ii) Target DNA sequence (e.g. GC content, long single- or di-nucleotide
streches) -Fig. 2
Fig. 2. Effect on accuracy of template sequence composition &
base range position from primer with automated computer base-calling.
(iii) Data processing (ie. manual vs. automated base calls)
-Fig. 3 (iv) Single- vs double-strand sequencing -Fig. 3 (v) Number of
independent readings at any one given position -Fig. 3
Fig. 3. Various approach to DNA sequencing significantly affect
error rate.
|
