You will need to know in advance which SNPs are already associated with control and with diabetes. If your primers are already designed around those SNPs then a common way to detect the SNP is to use allelic discrimination (TaqMan) fluorescent probes designed to span across the SNP. You need two probes - one for each allele of the SNP. Each probe has a different fluorescent color. Probes are often 16 nucleotides long with a fluor and a quencher and a minor groove binder (MGB).

Another method is high resolution melt, which can be done with only SYBR Green dye, but which requires precise temperature control of the thermal cycler. It relies on the slightly different melting profile of each allele.

Is this the information you were looking for?

Libby

Sequencing and analysis genotype (like phylogenetic tree etc)...

HI,

Well as mentioned, TaqMan based and Sanger based approaches are feasible. You might have an easy way out to do 'sequencing'. So PCR, cleanup, cycle sequence and Sequence. Both strand confirmation gives you an idea about your control and patient specific sequence and the SNP whether its Homozygous or Het.

You should see a peak under peak for a Het SNP and single peak for Hom. eg. G>A

If het will show you a G and A combined peak. If hom, then A peak single where your control is GG. genotypes can be either GG (Control), GA het (patient) or AA hom (patient).

Sanger is easy and clear.

Good luck.

Since you have a;ready designed the primer for amplification, u can genotype the samples using sanger sequencing. The length of the amplicon that can be sequenced depend on the machine available. for example if you have AB 3730 DNA analyser, u can sequence upto 800-1000 bases in one reaction. you can then align the sequences and look for variations. You can use Mutation Surveyor software to see if theres any variation in your sample sequence.

If you already know the SNP which you are looking for, u can see if theres any specific restriction site in the SNP site which can be used to identify the genotype using RFLP.

Other genotyping method include Taqman genotyping assay from Applied Biosystems and KASPar from K Bioscience. For both, u just need to give the rs id and they will give u primer set which can be used for genotyping through amplification in a Real time PCR machine.

Sequencing your PCR product will give you the most definitive answer.

DNA sequencing will provide the most comprehensive and clear answer.

Yea, if you have already designed primers, amplify that region, go for direct sequencing, align the received sequence against the wild type sequence and locate your region or that particular nucleotide. if you need details step by step, please do not hesitate to e mail me.

In my view the easiest way is to genotype the STR markers or SNPs flanking the region of interest. Go for the conventional Li-COR based system or the modern capillary eletrophoresis method. sequencing is expensive as compared to genotyping.

Thanks a lot- Elizabeth Rutledge , Kalpita Karan,Saradalekshmi K R, Eileen Roulis , Muhammad Alam, Noushad Karuvantevida ...it was very informative.Since there are about 200 samples so sequencing will be very tough.So i want to go for RFL and my SNP is already known but the problem is after digesting the amplicon with specific Restriction enzyme,how can we decide that say for G>A, two bands will be for GG and one band will correspond to GA or AA?

Dabhi, I will again suggest you to do STR genotyping. It is not very expensive as well as it won't give u hard time with experimentation. your choice now! good luck! if u need help with it. Im here

Thanks Beenish. Can you give me the detail of STR genotyping?

Sure Dabhi. I can send you smthng to read about STR genotyping, if you like

Quite an old paper, but for initial learning it is good. You can have an idea what I am talking about. And then about handling the experiment, I will help you as much as possible, in silico :)

Article Capillary Electrophoresis STR Analysis: Comparison to Gel-Ba...

thank you so much Beenish...I appreciate your efforts.It was very helpfull.

Welcome

Going back to the RFLP question. If you know what the SNP is, then you want to choose a restriction enzyme that would cut the A allele, but not the G allele. It also matters where the SNP is in the amplicon, because that will determine the size of the bands which are created. But when you run it on the gel you might see something like short band(s) - homozygous A, long band - homozygous G, both short and long bands - heterozygous.

thanks - Deborah Fenner and Andreas Moor

if you are interested in some specific SNPs I suggest you the TaqMan® SNP Genotyping Assays. You only have to know the rs number of the SNPs. You will need a real time thermal cycler. to read the plates

Thanks Vicent Guillem

If you know your SNPs then you can go for ARMS-PCR (Ye et al., 2001). Its a relatively cheap and feasible method. Or as others suggested go for high throughput methods few of them would be Sequenom genotyping (you can easily outsource these) and now there is also FLUIDIGIUM technology.

All the abovementioned techniques are useful only for detection of known SNPs. For novel SNPs sequencing or pyrosequencing are good options. Hope this helps.

You can use primers directed to you three genes labeled with different fluorochromes. Following PCR, the amplification products are diluted and denaturated with formamide. An internal size standard should be added

The samples can be run on an ABI 310 genetic analyzer (Applied Biosystems). For

accurate allele size determination, the sizes of the PCR

products can be determined by using GeneScan analysis software.

The alleles are then designated by their sizes (in base pairs) or by the number of repeats. Hope this help!

thanks a lot-Abhimanyu -and Raquel Sabino.it was really very helpfull.

or you could also to dHPLC technique...

dear colleagues I wonder, why don't you simply get the Amplicons sequenced, and analyse the sequence resutls by Bionumerics Software this is the easiest way based on bioinformatic methods ... we are doing the same procedures on Salmonella samples .... would be pleased to have your worthwhile viewpoints ... regards-rain