musiclover
Forum Junior
Topics: 13
Posts: 35
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Hi,
I don't understand part of this mol biol q'n from Kaplan - can anyone help me?
The question refers to sequencing a gene for haemochromatosis. The gel readout in provided and you have to work out the sequence of the gene. I can read the gel but I don't understand why the sequence of the gene is backwards as well as complementary to the sequence on the gel.
Question: A researcher wants to sequence part of the exon sequence of the HFE gene known to contain a mutation. Using a primer that binds to the 3' flanking region on the coding strand to initiate replication of the HFE gene and radiolabelled dideoxynucleotides to terminate synthesis, he achieves the results shown on the gel below.
The sequence, from top to bottom of gel is GACAGTAACGTCGT.
The correct answer is CTGTCATTGCAGCA.
Can anyone help?
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| bluedusk
Forum Elite
Topics: 35
Posts: 217
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Think of the correct answer string as a 5' to 3' sequence. If you were building a complementary strand from the 3' end (which is what you do in dideoxy sequencing), your longest chain would synthesize all the way to the 5' C, where it would terminate as a G, right? So the longest chain ends in the G column, and would be at the top of the gel. The next longest chain would do the same thing, but terminate at an A, right? So it would be a little bit shorter and migrate a little farther than the chain in the G column. And so on....
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| musiclover
Forum Junior
Topics: 13
Posts: 35
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thanks so much! now I understand
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| bluedusk
Forum Elite
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: ) good luck.
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| krishie
Forum Senior
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Posts: 128
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Please correct me if I am wrong.I thought that the template strand was GA---------GT.hence the gene sequence is complementary to the template strand,i.e-CT-------------CA.
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krish
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