Question: (Closed) How Is Epitope Mapping Done
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gravatar for WinnyH
6.3 years ago by
WinnyH0
WinnyH0 wrote:

I have question regarding Site-directed mutagenesis and Mutagenesis mapping. (See copied from Wikipedia below)

For site-directed mutagenesis how do you introduce systematic mutation of amino acids into a protein sequence? Is it through genetic manipulation through PCR? In Mutagenesis mapping where comprehensive mutation library is used. How is this comprehensive mutation library is made?

What's the difference between Site-directed mutagenesis and mutagenesis mapping.

Site-directed mutagenesis: Using this approach, systematic mutations of amino acids are introduced into a protein sequence followed by measurement of antibody binding in order to identify amino acids that comprise an epitope. This techniquedo can be used to map both linear and conformational epitopes, but is labor-intensive and slow, typically limiting analysis to a small number of amino acid residues.

Mutagenesis Mapping.[8] This approach utilizes a comprehensive mutation library, with each clone containing a unique amino acid mutation and the entire library covering every amino acid in the target protein. Amino acids that are required for antibody binding can be identified by a loss of reactivity and mapped onto protein structures to visualize epitopes.[9] This approach has recently been used to epitope map a panel of antibodies against human CCR5, a GPCR coreceptor for HIV entry.[10]

Thank you!

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ADD COMMENTlink modified 6.3 years ago by Devon Ryan95k • written 6.3 years ago by WinnyH0

As mentioned, this has nothing to do with bioinformatics, I just answered too since I'm familiar with mutagenesis.

ADD REPLYlink written 6.3 years ago by Devon Ryan95k
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gravatar for Devon Ryan
6.3 years ago by
Devon Ryan95k
Freiburg, Germany
Devon Ryan95k wrote:

This has nothing to do with bioinformatics, so I'll close the question after answering.

Regarding site-directed mutagenesis, yes, this is usually done with PCR. Just google "quikchange mutagenesis" for details. Mutagenesis mapping is actually pretty similar, it's just that the mutations are done randomly rather than in a directed manner. The general idea is to PCR a doubly epitope (e.g. with HA and V5 on opposite ends) tagged amplicon in conditions where the error rate of the polymerase is increased. The results are then normally sequenced, possibly after subcloning, and then the results pooled.

You actually could have found all of this by following the links on the wiki page.

ADD COMMENTlink written 6.3 years ago by Devon Ryan95k
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