Has anyone seen anything like this before during Sanger sequencing? We have a control sample that was run inline, and it was perfect, indicating the Sanger setup and instrument were performing optimally. Reinjection did not remove the interfering signal. The samples are from the following: RNA extraction -> cDNA Synthesis (using Oligo dT primer) -> PCR product -> PCR Cleanup (magnetic bead) -> Sanger reaction (BigDye v3.1).
THE NOISE OBSERVED IN YOUR CONTROL MAY BE DUE TO SEVERAL FACTORS EXAMPLE:
IF IT IS A PCR PRODUCT FROM BACTERIA USING PRIMER 16S, YOU MAY HAVE SEVERAL DIFFERENT PRODUCTS IN YOUR SAMPLE THAT ARE AMPLIFYING THOSE PRIMERS SINCE JUST AS THERE ARE UNIVERSAL PRIMER 16S, THE SPECIFIC ONES ARE NOT SO UNIVERSAL.
IF IT IS A VIRUS PRODUCT USING PRIMERS SUCH AS RVA AND YOU WANT TO SEQUENCE A VP7 USING PRESERVED PRIMERS, THERE MAY BE CO-INFECTIONS THAT HAVE MORE THAN ONE VP7 GENOTYPE AND FOR SUCH REASON YOU HAVE THAT NOISE.
CO-INFECTIONS AS WELL AS PROBLEMS IN THE PURIFICATION OF THE PRODUCT AS WELL AS IF THEY ARE PRODUCT USING VERY DEGENERATED PRIMER THAT ARE CAPABLE OF AMPLIFYING MORE THINGS THAN KNOWN.
THERE ARE MISSING MORE DETAILS ABOUT WHAT YOU ARE PROCESSING TO GET A CLEARER IDEA, WHEN USING OLIGOT FOR CDNA I ASSUME THAT YOU ARE LOOKING FOR MATURE mRNA BUT IT DOESN'T HELP MUCH.
Can you attach the original .abi sequence file please?
The early large peak indicates that the kit reagents have not been used as does the low signal intensity and the attempt by the analysis software to produce a signal where there is none. Some general reasons for this happening are
– Not enough template
Primers not annealing
Primers not at right concentration or degraded
Inhibition by impurities
Since other samples worked the primers are presumably good so it may be that this sample has mismatched base(s) near the 3' end of the primer or not enough template look like the more likely reasons for this effect
I have only seen this when ddNTPs were added separately and one of them was not diluted properly.
It may also be possible that you need to replace the bigdye mix or recalibrate the machine if this keep repeating.
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