Hello everyone,
I need your recommendation about the best method to detect variants for a specific gene in a big sample. let us say that I wanted to detect variants of CYP2C19 in 100 patients, would a PCR for a specific known mutation be the best way to do it or should I use another method? your suggestions will be appreciated.
If you are just looking for one known mutation then ARMS or ACRS/(DCAPS) ( one mismatched primer and restriction digest for the base change) are both good and cheap and handle large sample numbers but will not give any information about other mutations.. If you are looking for unknown mutations then sequencing cDNA from patient RNA is very efficient or simply amplifying each exon and sequencing from the DNA sample. NGS is excellent but still expensive.
If your disorder is associated with one major mutation and other mutations on rare occasions then USE ARMS or ACRS pcr to sort out the majority of samples and sequence the remainder
You need to do the multiple sequences alignment (MSA). Sequence you target gene from each sample and proceed to MSA using an appropriate software: MEGA, BioEdit, etc.
I suggest for gene mapping using microarray either on Illumina platform or Affymetrix platform, particularly for such large sample volume
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