Hi Varsha. The additional details help. So you are trying to design an RFLP (restriction fragment length polymorphism) assay to detect a point mutation in EGFR and checking if exon 14 is present/absent in MET. So I have a few suggestion and comments. For both genes, I am assuming you have or will design PCR primers in order to amplify the region of interest for either gene.

I did a quick search for MET, so I could be mistaken, but exon 14 seems to be 141bp long. This means that for MET, you could simply run a PCR on cDNA with one primer in exon 12/13 and one in 15/16. If you have a control sample, where you know exon 14 is present, you would be able to run the both the control and your sample of interest on an agarose gel (without doing any restriction enzyme digestion) and see if the product in your sample is at the same position as your control containing exon 14, or if it is lower on the gel by ~140bp. You would need a 'high' percentage agarose gel, at least 2% to resolve that size (see: https://www.promega.ca/resources/pubhub/enotes/what-percentage-agarose-is-needed-to-sufficiently-resolve-my-dna-sample/)

For your EGFR point mutation, you would also design PCR primers to amplify the sequence of interest. The consideration here is also the fragments it generates as you will want to make fragments large enough to distinguish after on an agarose gel. Ideally the RE would cut once or not at all depending on the presence or absence of the point mutation in question. You can use the NEB cutter tool that I mentioned previously to do find a RE for your point mutation. Hopefully one exists.

There is also consideration of the MAF (minor allele frequency) of your point mutation. I don't know the details of your particular case but the point mutation may be homozygous/heterozygous and depending on your sample source there could be the possibility of normal DNA presence. This means your point mutation may be present in very low % compared to the normal DNA. If it is a possibility in your case, you may want to run a dilution series to see at which MAF you stop detecting the point mutation. Take a look at this thread here. I show an example of testing an RFLP for a gene with varying mutation frequency (https://www.researchgate.net/post/Could_you_help_me_with_the_quantification_of_minor_variants-Sanger_sequencing)

Good Luck!

I am not entirely sure what you intend to do with the restriction enzymes in your study. You would need to provide more specific details of what you hope to achieve with the restriction enzymes to get a more detailed answer. However, a great tool for choosing restriction enzymes is the NEBcutter. You paste the sequence of DNA in FASTA format that you need digested and there are a few options that you can select to get results more suited to your needs. You can find the tool here (http://nc2.neb.com/NEBcutter2/)

Hi Pawel Buczkowicz,

Thanks for your reply.

My aim is to check exon 14 deletion in cMET gene. I want to cut my DNA with such restriction enzymes which will cut my target sequence(i.e exon 14 sequences). If chosen enzyme is cutting my target sequences then it means exon 14 is present in my target sample and it will  show 2 or more bands (according to enzyme) in gel. If my target sequences is absent in my sample then restriction enzyme would not work and single band will appear in gel.

Secondly for studying point mutation:  Since restriction enzyme are specific they would not cut mutated sequences (example if CTG is converted to CCG) so on the basis of gel I can discriminate between mutated and wild type.

I want to know how can I choose restriction enzymes specific for target sequences? 

Hi Varsha. The additional details help. So you are trying to design an RFLP (restriction fragment length polymorphism) assay to detect a point mutation in EGFR and checking if exon 14 is present/absent in MET. So I have a few suggestion and comments. For both genes, I am assuming you have or will design PCR primers in order to amplify the region of interest for either gene.

I did a quick search for MET, so I could be mistaken, but exon 14 seems to be 141bp long. This means that for MET, you could simply run a PCR on cDNA with one primer in exon 12/13 and one in 15/16. If you have a control sample, where you know exon 14 is present, you would be able to run the both the control and your sample of interest on an agarose gel (without doing any restriction enzyme digestion) and see if the product in your sample is at the same position as your control containing exon 14, or if it is lower on the gel by ~140bp. You would need a 'high' percentage agarose gel, at least 2% to resolve that size (see: https://www.promega.ca/resources/pubhub/enotes/what-percentage-agarose-is-needed-to-sufficiently-resolve-my-dna-sample/)

For your EGFR point mutation, you would also design PCR primers to amplify the sequence of interest. The consideration here is also the fragments it generates as you will want to make fragments large enough to distinguish after on an agarose gel. Ideally the RE would cut once or not at all depending on the presence or absence of the point mutation in question. You can use the NEB cutter tool that I mentioned previously to do find a RE for your point mutation. Hopefully one exists.

There is also consideration of the MAF (minor allele frequency) of your point mutation. I don't know the details of your particular case but the point mutation may be homozygous/heterozygous and depending on your sample source there could be the possibility of normal DNA presence. This means your point mutation may be present in very low % compared to the normal DNA. If it is a possibility in your case, you may want to run a dilution series to see at which MAF you stop detecting the point mutation. Take a look at this thread here. I show an example of testing an RFLP for a gene with varying mutation frequency (https://www.researchgate.net/post/Could_you_help_me_with_the_quantification_of_minor_variants-Sanger_sequencing)

Good Luck!

 Thanks Pawel

It's very helpful.

I want to know for studying Point mutation in EGFR should I use gDNA (double stranded) or cDNA?

For exon 14 skipping in cMET gene (is post transcriptional change) I assume cDNA should be used then gDNA. 

Can you help me in this?

Thanks

Whether you use cDNA or gDNA for the point mutation depends on what question you are trying to answer. If you are looking only to see if the point mutation exists in the genomic DNA of your sample then you can do gDNA, but if you want to determine whether the EGFR with the point mutation is being expressed (at the mRNA level) then you would want to use the cDNA. The possibility being that the point mutation exists as heterozygous mutation in EGFR but that the wild-type allele is the only one being expressed, or vice-versa or something in between.

Yes, if the cMET is an exon skipping event during transcription then use cDNA for that experiment. To verify this experiment you could also do a Western blot for cMET to see if the you are getting a shift related to having a lighter isoform of cMET due to skipping exon 14. It also seems that the exon 14 skipping in cMET is a result of a splice site mutation (https://www.ncbi.nlm.nih.gov/pubmed/27223456) which you could test for as well...possibly by Sanger sequencing.

Thanks a lot Pawel

Very helpful...

Restriction Fragment Length Polymorphism (RFLP) analysis

RFLP refers to heritable differences in the length of DNA fragments from the specific region of DNA generated by restriction enzymes due to DNA sequence differences.

Determine the mutation that you wish to investigate and design genomic primers for amplification of the required DNA fragment by isolating the section in the gene that you are interested in.

Single Nucleotide Polymorphisms (SNPs)

SNPs are point mutations caused by substitution, insertion or deletion of one nucleotide. 90% of the variation in the human genome is due to SNPs (Brumfield 2003) In order to determine the level of variation in the population, sequences of different individuals were compared by first amplifying the fragments using PCR and then by detecting differences using direct sequencing, RFLP or melting point differences.

Known homozygous samples for the allele being investigated must be included in each enzyme digestion to ensure that an efficient digestion has occurred.

Restriction enzyme digestion (RFLP)

Background:

RFLP is a method designed to detect genetic variation and involves the cutting of DNA with restriction endonucleases (enzymes that cleave DNA at sites specifically related to nucleotide sequences). The resultant fragments of DNA were separated on agarose gels and scored as to the presence/absence of a band of a particular size.

There are several hundred restriction enzymes available for use today, all of which differ in recognition and digestion sites. The method requires a relatively large amount of DNA to be successful and the DNA being analysed must be of a high quality.

After amplification DNA must be digested using 10-20 units of the required enzyme, buffer and BSA in 1.5ml eppindorf tubes at the required temperature for each enzyme in a water bath.

The enzymes were specifically chosen to recognise a restriction sites that coincided with polymorphic variations in the gene undergoing investigation.

I hope that this is not too simplistic but it is the method that I used. I also have methods for cDNA if it helps.