If there is no PCR bias for the amplification of the one allele over the other allele, the heterogeneous point mutation should show up as an overlapping peak of G and A of about half the 'normal' height.

See an example here:

https://figshare.com/articles/_Sanger_sequencing_of_IFITM5_identified_the_identical_heterozygous_c_14C_T_mutation_black_arrow_in_all_the_affected_patients_with_IO_type_V_/777500

If you would like to have 'clean' sequences (only showing the G or the A), you should clone your PCR product in a PGEM T-easy vector or similar, and send individual clones for sequencing.

yep

If there is no PCR bias for the amplification of the one allele over the other allele, the heterogeneous point mutation should show up as an overlapping peak of G and A of about half the 'normal' height.

See an example here:

https://figshare.com/articles/_Sanger_sequencing_of_IFITM5_identified_the_identical_heterozygous_c_14C_T_mutation_black_arrow_in_all_the_affected_patients_with_IO_type_V_/777500

If you would like to have 'clean' sequences (only showing the G or the A), you should clone your PCR product in a PGEM T-easy vector or similar, and send individual clones for sequencing.

As Stated yes

Daan's answer is excellent

I would add to that by saying that you need to make sure your raw data strength is good and therefore that your signal to noise in sequencing ladder is high, i.e you have strong specific peaks with very little underlying noise: In that case your 2 bases will be distinct as they will have a different physical size

In the past having looked for SNPs in PCR amplicons by Sanger sequencing I would say it is straight forward if there is no background noise but more difficult where raw data is less strong and hence background noise can sometimes obscure an additional second specific peak: I would also suggest sequencing in both directions; that is using both your forward and reverse primer (from your PCR amplification) for Sanger sequencing. That way you can be absolutely confident you are seeing a genuine base substitution

Another point I will add is that if you expect to see a base substitution, try and deliberately design your primers so that your anticipated base change is 100bp to 200bp down stream from your start site. That way you will avoid complications with inefficient recovery of the first 50bp of your sequencing ladder - which tends to happen - and also large amorphous blobs called 'dye blobs' (caused by aggregated dye terminators, particularly in old big dye mix), both of which will obfuscate your visible base change

If incidentally you are required to place primers such that your expected base change does indeed lie within the first 50bp then

A) Use of big dye removal columns will minimise those amorphous blobs

B) Alternatively, if you clean up your Sanger sequenced amplicon by ethanol precipitation, Wash your precipitated pellet 2 x with room temp 70% ethanol and also when you initially precipitate with Ethanol and salt (sodium acetate) add 1/10 x vol of 1mM MgCl2 as well: This selectively precipitates nucleic acid products under 100bp and will increase your signal to noise in this region of your read  

Many thanks Joas, Daan and Laurence for the explanation. I really appreciate it.

I am struggling with the CLC Main Workbench to see the peaks. I can't get it to show me the peaks. Is there an alternative (free) software you guys could suggest to visualize the peaks?

Thanks, again,

Ariadna

I am using a program that is called SnapGene, which works well for visualizing, but doesn't allow me to export the pictures (then I need to pay).

Keep in mind that not all sequence file-types contain the information about the peaks. Your sequencing service probably provides different types of files with the results. The files that contain the information about the peaks (the actual chromatogram) will most likely have the file extension .scf or .abi/.ab1/.ab

Now this makes sense!!! Because I was using the wrong files for it in CLC!!! Thanks so much, I'm new to this. I appreciate it, Daan!!!!!!!!!! So much! Thanks!

good that your problem is solved Ariadne. Possibilities re your question about free software for viewing sequences there are many programs. I have used Chromaslite, ABI have a free program which gives lots of information about the quality of your electropherogram called Sequence scanner and the trial version of Gene Code's software Sequencher is excellent but you cannot save,print or export the aligned sequences but is good for aligning sequences with other sequences or reference sequences

Thank you, Paul. I stumbled across a few while searching (e.g. 4Peaks). I will note down the ones you suggest for the future. Again, many thanks!