CEQ-A-0003
A PPLICATION I NFORMATION
Genetic Analysis: CEQ™Series
USE OF BETAINE TO PROMOTE READ THROUGH
OF PREMATURE STOPS IN DNA SEQUENCING
TEMPLATES
CEQ 2000, CEQ 2000XL, CEQ 8000 & CEQ 8800 DNA Sequencer
Joanna Craggs PhD1, Lee Edwards PhD2 and Jim Thorn PhD1
1 European GA Applications Support Team
2 UK GA Sales Team
Beckman Coulter Europe
Notes
The WellRed dye terminator sequencing chemistry used in the CEQ series of DNA sequencers is extremely
efficient at resolving difficult templates such as those that are G/C or A/T rich, contain microsatellite repeats
or poly(A)/(T) regions. However, some DNA templates form hairpin structures that cannot be resolved by the
standard sequencing protocol. These typically present as a “stop” in the raw data before the expected end of
the template.
Betaine is routinely used to improve PCR reaction efficiency through regions of strong secondary structure.
It works by destabilizing GC basepairs and improving the processivity of the DNA polymerase. This reduces
"pauses" in polymerization caused by secondary structure that can induce the polymerase to disassociate
from the DNA strand. Betaine is effective at final concentrations between 0.7 M and 2.0 M.
Primer binding Tm's will be effectively reduced by betaine and consequently primer annealing temperatures
should be reduced by 1-2 °C. One concentration of betaine may work well for some templates but not work
for others so a bit of titration and experimentation is necessary for optimal results.
The work described in this Note is for guidance only and has not been fully validated.
Applications Team Europe
Page 1
Jim Thorn
A PPLICATION I NFORMATION
Genetic Analysis: CEQ™Series
Protocol
Reaction Set-up to Optimize Pre-Heat Treatment
1.
a.
2.
3.
4.
5.
Make DNA to final volume of 6µl in water.
Use 50-100fmoles Plasmid or 10-25fmols PCR product.
Heat to 96 °C in a thermal cycler with heated lid.
Set up five reactions for the same sample with heat treatment times from 1 to 5 minutes.
Add 3.2pmoles in 2µl primer and 8µl Quickstart mix.
Add 4µl 5M Betaine (Sigma Part B0300 for 1.5ml of 5M solution).
Altered Thermal Cycling Conditions
1.
2.
Adjust Thermal Cycling Conditions as follows:
Hot start
96 °C 2 minutes.
3.
Followed by thirty cycles of:
a. 96 °C for 30 seconds.
b. Primer Tm - 5°C for 20 seconds
i. Do not use an annealing temperature below 50°C, because low annealing
temperatures may cause mis-priming.
c. 60°C for 4 minutes.
4.
5.
6.
7.
Then hold at 4°C until ready to clean up the reaction.
Use standard Ethanol or spin column/plate method.
Resuspend in 40µl SLS in the sample plate and overlay with mineral oil.
Run on standard CEQ protocol to achieve desired read length.
____________________________________________________
Applications Team Europe
Page 2
Jim Thorn
A PPLICATION I NFORMATION
Genetic Analysis: CEQ™Series
EXAMPLE RESULTS
Figure 1 Raw Data Traces from Quickstart and Quickstart with 1M Betaine
Quickstart
Quickstart + 1M Betaine
Applications Team Europe
Page 3
Jim Thorn