first of all do you know where the mutation occurring? just guess or any idea, in which gene/s? if you do not have any information of gene and mutation, i would start with Sybr green qRT-PCR where you can test many genes at same time and its relatively cheaper. once you have one or two candidate targets you can switch to Taq-man based qrt-pcr and get more quantitative and qualitative answers.

Dear Vrutant

Thank you very much. In fact, I want to consider a region about 10 kb in intronic region which is including 3 hypersensitive sites. I found some papers for using qPCR. However, I need more information. I really appreciate it if you provide me some practical protocols or useful papers. Thanks in advance.

Cordially,

Hadi

to identify the intronic region and sites of mutation you will need to design proper primers which can detect small changes. protocols and other related stuff you can get from vendor's website. which real time pcr machine do you have? we usually use ABI step one system and also reaction mixtures from them.

http://www.lifetechnologies.com/us/en/home/life-science/pcr/real-time-pcr.html

this link will provide you more information about real time pcr.

i hope this will help you.

vrutant

I really appreciate your kindness. Have all the best.

hi hadi.

I think HRM Real - Time PCR will be useful for your study, particularly if you know the bases that replaced or inserted/deleted in slightly sequence.

regards

You could also consider sequencing.

Hi Hadi,

As Vrutant mentioned, I recommend you to start with SYBR green method to identify your mutation´s regions. Then you can design specific Probs for those mutations. In your case primer design is so important. They must be located in the region with high risk of mutations or maybe you have to do DNA walking. Maybe these papers can help you in unknown deletions identification. “Detection of unknown deletion in beta-globin gene cluster using relative quantitative PCR method” and “Development of a quantitative real-time PCR assay for detection of unknown alpha-globin gene deletions”

Good Luck

Dear all,

thank you to your scientific supports and immediate attentions. Dear Somayeh I have another big thanks to you. It is exactly what I've needed.

Cordially,

Hadi