How to read tricky electropherograms?

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    • #10575
      Jamus
      Participant

      Dear all,
      (Please see attached file). I am having complications with reading electropherograms. In the attached file, how do you decide on what base it is and what makes you think so?

      What does it mean by "heterozygotes" in the context of electropherograms?

      How does that arise?

      Thank you.

      Regards,
      Jamus


      Attachments:

    • #87645
      wbla3335
      Participant

      Your examples could indicate background noise or double signals. If a nucleotide site is heterozygous, you will see two peaks, ideally of equal height but not necessarily so, representing the presence of two real signals. Background noise, however, is common. If your sequence is generally clean and you come across sites as in your examples, I would suspect heterozygosity at both sites. Ideally, it is best to have known homozygotes as controls for interpreting such data.

    • #87659
      Jamus
      Participant

      Thanks for the reply. How can a nucleotide site be heterozygote (i.e. have two bases at the same time)?

    • #87660
      wbla3335
      Participant

      Depends on what you’re sequencing. Clones, of course, are (should be) always homozygous for all positions. But if you PCR genomic DNA from a diploid organism and sequence the PCR product, you will get double peaks at all sites that are polymorphic. If your primers aren’t specific for one gene of a gene family, you can amplify more than one gene and get multiple peaks for each site that differs in the different family members. The possibility also exists that you could get three or more peaks from polyploids.

    • #87687
      Jamus
      Participant

      Yeah, I am doing a PCR genomic DNA from a diploid organism i.e. Human.

      I think I require some basic explanations in the first place. How can polymorphism arise in such a way that TWO nucleotides occupy the same spot?

      Thanks.

    • #87689
      wbla3335
      Participant

      Human cells have 23 pairs of homologous chromosomes. 23 come from the father and 23 come from the mother. So all genes occur in two copies within one individual. These copies are known as alleles. The version (allele) of gene A that came from the father may be slightly different than the version of gene A that came from the mother (gene A is heterozygous). Both versions, though, occur in (almost) all cells. If you PCR this gene, the PCR product will be a mix of both alleles. If you then sequence this PCR product, you will get double peaks at all the nuclecleotide sites that differ between the mother’s and the father’s alleles because both alleles are in the PCR product. OK?

    • #87704
      Jamus
      Participant

      Thanks for the prompt reply 🙂

      In the diagram attached, on one particular gene, the alleles from the parents of a person is different at that particular point. Which bases are detected in the electropherogram?

      If you are finding my questions too "frustratingly" to answer, could you please refer me to a website for more details?

      Thanks 🙂


      Attachments:

    • #87705
      Jamus
      Participant

      Let me guess!

      On the forward strand, the forward primer will sequence both the top DNA chain of the "Mom" and "Dad", and that’s why you get "A" and "G" peaks in the electropherogram for the forward strand?

    • #87712
      wbla3335
      Participant

      Right!

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