Troubleshooting your sequencing results
Below are guides to frequent problems associated with Dye terminator Sequencing and there potential solutions.
There are many variables associated with DNA sequencing that can affect data quality.
Inadequate template concentration. (The most common reason) Templates that are too low in concentration will all generate sequences low in signal intensity. When signal intensity is low the analysis software has difficulty in resolving the base peaks from background noise hence poor base calling and poor data quality. On the other hand, highly concentrated plasmid preparations can have a highly detrimental effect on the electrophoresis of sequencing reaction product. They cause the capillary to become blocked, inhibiting the current and causing the reaction product to pass through the capillary slower than normal. Detection of these products begins later, resulting in poor sequencing results.
“Dirty” templates, contain impurities which can inhibit the Taq polymerase activity or/and prevent the electrophoresis to be performed successfully. Common contaminants are:
- Salts (EDTA, NaCl,NaAc,Kac,KCl)
Note: For sequencing, DNA should NOT be dissolved in TE buffer because of EDTA's ability to bind Mg++ which is critical to Taq polymerase activity.
- Phenol quenches fluorescent dyes
- Also proteins, detergents (SDS, Triton X-100), RNA, chromosomal DNA, organic chemicals (ethanol, chloroform, phenol), divalent cations (Mg, Ca,Mn),excess PCR primers, dNTPs, enzyme, and buffer components from PCR
Low primer concentration. Primer concentration should be at least 10pmol/ul
Primer quality: Primers should have most of the following characteristics to be able to produce good, consistent sequencing data.
- High purity
- No mismatches
- No potential alternative binding site in the template
- No secondary structures present, especially at the 3' end
- 1-8 bases minimum, longer for AT rich primers
- Avoid runs of more than 4 of the same bases
- Tm adapted to our annealing temperature (50°C)
Our primer/template annealing step occurs at 50°C. Thus, if your primer Tm is much lower than 50°C hybridization to its complementary template will be much less efficient and a lesser number of extending fragments will be generated. Increase your primer Tm by adding additional bases to the 5’ or 3’ end to raise the Tm to be within the range of 52°C-58°C. Degenerate primers and those with mismatched bases will also show decreased hybridization efficiency due to reduction of the stability of primer binding, and if degeneracy or mismatches occur at or near the 3’ end of your primer, it is highly likely that your sequencing attempt will fail.
Difficult DNA content: If your DNA template failed to give good sequence data with the standard BigDye Terminator chemistry and the failure is not due to the poor template/primer quality, incorrect quantity or other improper sample preparation steps, then the DNA may be a Difficult Template containing one or more of the following:
- AT or GC rich stretches
- Secondary Structure
- Repeats
- Homopolymer regions
- Cosmids, P1 clones
The DNA Sequencing laboratory does have alternative protocols and reagents that might help alleviate some of these issues.