Dear Sir/Madam,
I am working on DNA sequence (Sanger's method) to find the SNP's for my Project work. I suspected that one of the mutation is novel, how could I prove that as a novel mutation for publication.
what should I do now for confirmation?
Is their any alternative technique for prove?
Shall I go through RFLP? is it enough to prove or else?
you should run normal samples and show that it is not found in about 100 other samples if in the human genome. Also look at the physical properties. Does it change a splice site,cause a stop codon,change a disulphide bond, change properties like small size or polar or aliphatic to large ,non polar or aromatic any of which might change the shape and structure of the dna Consider also if it is in an important part of the protein...a binding domain for other proteins or dimerization region for instance Check also that it does actually cause a change in amino acid.
I do not think that restriction digestion is necessary but sequencing in both directions is the very best evidence for the existence of a sequence
look in SNPdb and other SNP databases and your mutation is not listed than it is a novel mutation.
Dear Abhishek kumar Sir,
If it is a novel mutation, when we submit the paper to publication they may ask for confirmation so which technique will be used for reconfirmation?
you should run normal samples and show that it is not found in about 100 other samples if in the human genome. Also look at the physical properties. Does it change a splice site,cause a stop codon,change a disulphide bond, change properties like small size or polar or aliphatic to large ,non polar or aromatic any of which might change the shape and structure of the dna Consider also if it is in an important part of the protein...a binding domain for other proteins or dimerization region for instance Check also that it does actually cause a change in amino acid.
I do not think that restriction digestion is necessary but sequencing in both directions is the very best evidence for the existence of a sequence
One thing you can quickly do is to run nucleotide BLAST, see whether it is matched (but not 100%) with the wild-type gene you are studying. If it is a somatic mutation, you can check on COSMIC (Catalog of Somatic Mutations in Cancers) database (click links below). Last possible way is to key in what exactly your finding on Google and see whether you can find a matching result. E.g. Let say you found a mutation on KRAS 35G>T (mutation at KRAS gene, sequence # 35, Guanine to Thymine substitution), type that on Google. That was a known SNP and you would find plethora of reports on that (just an example for explanation sake). Good luck!
http://cancer.sanger.ac.uk/cosmic
https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq
Assuming the DNA is human, first BLAT your sanger sequence against the hg19 human reference and work out the chromosome and locus position from where it mismatches in the "details" link of the results (Blat is under the "Tools" menu")
http://genome.ucsc.edu/
Then go back and view the position in the "browser" link. Scroll down and ensure that under the Variation section "All SNPs(147)" is set to "full" then click refresh. Zoom in to the locus and see if there is a dbSNP ID shown at that position.
Then also check all of the following websites to see if the same variant is listed:
http://exac.broadinstitute.org/
http://gnomad.broadinstitute.org/
https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/
http://evs.gs.washington.edu/EVS/
http://igm.ucsd.edu/gme/data-browser.php
http://cancer.sanger.ac.uk/cosmic
Hi,
First check its frequency in online data bases, such as
http://exac.broadinstitute.org/
https://www.ncbi.nlm.nih.gov/variation/tools/1000genomes/
http://evs.gs.washington.edu/EVS/
If it's not found in the normal population then go for the mutation prediction tools,
http://www.mutationtaster.org/
http://sift.jcvi.org/
http://genetics.bwh.harvard.edu/pph2/
http://mutationassessor.org/r3/
http://cadd.gs.washington.edu/
If any 3 of these suggest that your SNP is disease causing.... Then its confirmed..!!!!
Thank you.
Best wishes,
Umair
Hi yedu. For that you first have to check it through ensemble database, once confirmed that its a novel mutation, try finding its minor allele frequency through EXAC database. You can also go for its insilico analysis through different softwares like SNIF, Polyphen2 and mutationtaster. best of luck
A simple safe and easy way for your case is, try to register your detected SNPs in European Variation Archive (EVA) if the variants are belong to non-human organisms or in NCBI for human. They just register the novel SNPs.
Good luck
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