While using Taq Polymerase for Sanger sequencing, does the addition of an extra A at the very end have a role to play in this observation ?
And if so, why is the large peak observed only at the very end of the sequence ?
I am sure that you are right that your polymerase used for making the pcr product is one of those that adds an A overhang. Then if you sequence from the other end you will get dye deoxy terminating at the last A by incorporation of dye deoxy T. All of the other deoxy T will also terminate here but these will not be visible as they are not dye labelled so I do not understand why the intensity is greater than the other bases which will have sequenced normally
Thank you Paul!
Going by your explanation, I believe my question may possibly be incorrectly phrased - rather than there being a large peak of T in the chromatogram, there would be a large accumulation of T in general at one end of the products due to the reason you've mentioned.
I am also interested to know whether there could be any possibilities other than this ?
Thank you!
Can you attach a .abi sequence file so that we can see this effect please Sreepadmanabh Mahadev because it does seem an unusual effect. If the sequence were very long or the sequencing mix over diluted and the near end of the sequence was TCG rich then it may be that these bases are getting depleted so relative to the ACG bases the T bases at the far end would be higher intensity simply because there is more dyedeoxy T remaining in the reaction mix
thank you
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