If I well understand, there is ONE "complicated" gene with mutations causing disorder. It is correct? What is the length of the complete gene (exons + introns)? In fact, some time, it is a single mutation in an intron which cause the disorder : http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2855871/

Restriction Fragment Length Polymorphism is always convenient, efficient and economic way to screen out mutations. Try it please. Hope you will get satisfactory results.

Thank you all for the answers.

Dear Antony,

it is long gene that contains 36 exons.

the associated mutation differs from population to population

the mutation is expected in more than 100 patients in a population that was not studied before; i.e. I need to screen all the exons in the 100 patients.

Dear Antony,

you have mentioned that sometimes it is a single mutation in an intron that causes the disorder , in this case we can not explain it as a product of translation ( silent, mis-sense, none-sense, etc.); then how can we explain a mutation in an intron????

Hi The best way to screen for mutations remains the Sanger direct sequencing

Hi, 

if your gene has some highly frequent mutations causing the disease, as I understood, I will first screen my population for them. I don't know if they are a lot, but surely less than 36 amplicons, and hopefully you can test some or all of them by RFLP. Once you discard all the patients carrying these frequent mutations, you will have less samples to screen the entire gene.

hope it helps,

bàrbara.

Depending on the equipment you have in the lab, you could try High Resolution Melting; Benchtop NGS; Sanger sequencing, in this order of convenience and costs.

In this case Sanger sequencing is the best choice. First sequence the hot spots and then gradually move to the less reported regions on the gene.

If cost and equipments (like Sanger sequencer, NGS etc) are limiting factors, I suggest doing screening for unknown mutations by techniques like SSCP, DGGE etc. (do both as they can complement each other specially for hot spot exons) and detection of known mutations by  PCR-RFLP (if applicable), ARMS-PCR and melting curve analysis (if Real Time PCR machine is available).

Sanger sequencing is always standard method for detecting and confirming mutations, even if after screening by NGS.

According to our market study, indeed, 70% of users is validating their NGS variants with Sanger sequencing. Our new UGent spin-off company pxlence is offering PCR assays for the entire human exome (https://www.pxlence.com).