Hello everybody.
For my project i need to identify the APOe isoforms present on my patient derived lines.
My idea was to extract DNA, amplify with PCR and send it for Sanger sequencing - basically how routinely done in multiple papers.
So far i used multiple primers (both designed by me or found in publications) that either target the whole area of interest where the mutation is (112 and 158) or singularly each of part - with zero results. In some cases the primers were working properly (showing a perfect single band on gel), but then the sanger sequinning came back extremely dirty and unusable.
I also tried multiple mixes of primers, PCR protocols, TAQs ecc, but all of them without any success or with same Sanger messy results.
Just as an additional information, the area of interest is extremely GC rich (70-80%).
Can someone advise or share a good set of primers that are tested and a related PCR protocol? if this won't work, we would proceed with exons sequencing, but this is time consuming and expensive.
Thanks a lot
Francesco
Are you telling the sequencing company that these are high GC samples?. What pcr purification method are you using or are you leaving this to the company?. You would certainly get better results if the plasmid were linearised by cutting enzymatically and there are additives like dmso and betaine and deaza analogues of the G base but commercially these are probably not available
Paul Rutland Hi Paul, thanks a lot for your answer.
We sequences with eurofins and asked for the GC rich analysis specifically. The samples were sent both as purified or unpurified (and then purified by the company), but the results were similar. The PCR mix includes also 10% DMSO.
Throwing the kitchen sink protocol.
20 ul reaction, DNA 0.25ug/ul 8 ul, Primer 15pmol 1ul, BigDye 6ul, GTP 2ul, Betaine 4 ul, Buffer 0ul
Cycling Conditions - 98C/10 min - 1 cycle | 98C/20 Sec, 55C/15 sec, 60C/4 min - 99 cycles | Hold 4C
Dear Francesco
There are tings to try but my answer depends on either having access to a sequencer or being able to run your own sequencing reactions and sending the product to Eurofins just for running on the sequencer. Everything I suggest is designed to minimise secondary structure in both template and sequencing primer. Essentially I agree with Kevin Cavallin Kevin about many changes.
So if your pcr primers work at 60c then let each one anneal at 60 not the 50c recommended by the ABI manual.
Choose a physical method of removing primers from the pcr but not exo sap which can fail if the primers have hairpins or G quadruplex structure.
In crease the denaturation time.
Work with linearised template
Extension temperature 64c not 60c
Add dmso to 5%
Separately add betaine to a final concentration of 1 or 1.5Molar
Add dmso and betaine together as above
Remember sequencers load samples electrokinetically so when using betaine be especially careful to remove all salt (betaine) before sending the samle for running on a capillary system
Sequence from both ends separately because the single strands will have different secondary structures.
Kevins protocol with both Bd3.1 and dGTP v 3.0 in a 3:1 mixture has been reported to work elsewhere and looks good
Concerning the amount of template for a sequencing reaction I would keep it low.....for instance You should aim to use no more than 25 ng of template times the length of the template in kilo bases. For example, if you are sequencing a 3 kb plasmid with a 2 kb insert, you should aim to use 25 ng × (3 kb + 2 kb) = 125 ng in total.
best wishes
Paul
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line