The gene frequency would be low say 2% to 10% in your population. That's why you are not finding the homozygous variant. What is the sample size for your patients and controls ?

110 patient and 61 control

my studying about breast cancer and my colleague also use RFLP to detect SNPs in diabetes patient and also the same state does not have any homozygous genotype

Dear Zinah,

It is possible. Some SNPs have low heterozygosity and as a result you will observe one of the homozygous genotypes in a low frequency. I suggest you to search the SNP ID (rs#) in the SNP database of NCBI and check its heterozygosity. Also, you can assess deviation of the SNP distribution from Hardy–Weinberg equilibrium which help you as well.

Yours,

Heidar

Dear Heidar

Thank you I will take your advice ,but maybe this related with Loss of heterozygosity (LOH) or this another thing!

grateful

Regards

If you need more assistance, please let me know the SNP you are genotyping.

Dear Haider Yes pls actually i need more assistance would you please find this link http://www.ncbi.nlm.nih.gov/snp/?term=4986826

and tell me what is the next step!

thanks alot

The NCBI link you shared showed the MAF, or minor allele frequency, to be 0.022 (2.2%). That means the frequency of the homozygote (q^2) will be 0.022*0.022 = 0.000484. ie. very rare, at least from the NCBI data. What are the frequency of heterozygotes in both of your datasets?

dear Stephen Doyle thank u for response i don't do the statistic analysis yet but Almost 40% of all samples are heterozygotes

Hi Zinah,

I checked the allelic frequency of your SNP of interest and the MAF is quite low across different population cohort. As they have also not found the homozygous variant in 100-200 samples (http://www.ncbi.nlm.nih.gov/projects/SNP/snp_ref.cgi?rs=4986826). Basically, the SNPs who have their MAF

Dear Anshika ,

thank youvery much for your assistance ,but what they meant by "FWD" RefSNP Alleles:A/G (FWD) is a abbreviate for any words? and would you please send to me the link of Quanto program software.

Grateful and Regards

The minor fequence allele usualle don´t appears as homozygous. You can create your positive contro with PCR strategies (mutated oligos) to discards false negatives.

Dear Ghazi,

I read most answers that appear evident to check and I complete to genotype some samples by sequencing to generate a positive controls for 3 genotypes.

Good Luck

Dear Zinah

Above comments are up on confirmation of your method part

If you have 40% heterozygote frequency, it is not normal to 0% homozygote frequency

Please describe your detailed method (enzyme, times …)

You should have suitable sample size (no less than 70-80 subjects)

You should have a single confirmed band in PCR

Your digestion step should be confirmed using positive control (you can use a suitable plasmid and check complete digestion of enzyme)

Digested bands should be in appropriate place (correct size)

40% digestion and no homozygote may suggest

1. star activity and false extra digestion

2. incomplete digestion of homozygote genotypes and wrong interpretations

Sequencing of samples is a good idea

(You know that other mutations in recognition site of enzyme will prevent digestion too)

But you need to confirm your method first

Regards

It is not just common, but very routine....

Hello, homozygous genotype in the PCR-RFLP technique, isn't uncommon,i don't have this cases you explain.check your protocol of PCR-RFLP technique and sample you use for experiment

Thank you very much all for your response

Dear Daneshmandi,

thanks a lot for very good comments :

first i have suitable sample size (about 200 subjects) till now ..

Regarding to the point of non-complete digestion actually I'm afraid that is the reason but i used enough amount ,10 units of enzyme and incubation time (1 hr- overnight ) and give the same result except of the bands seems weak in each incubation time more than 4 hours .

In general (HincII supplied by New England Bio labs ) the company recommend that 1 unit of restriction enzyme will completely digest 1 μg of substrate DNA in a 50 μl reaction in 60 minutes .

and actually i only use PCR product without digestion as a control i don't use a plasmid simply i don't know how!

finally i will send some of the samples to sequencing i think it is the best idea to conform the result grateful for all advices in deed

Best Regards

Zinah

Dear Zinah

Your sample size is suitable

As company recommendation “Enzyme volume should not exceed 10% of the total reaction volume to prevent star activity due to excess glycerol”

You should not use more than 5 micro liter of enzyme in 50 micro liter of final reaction, and your bands will be degenerated by star activity of enzyme.

Sequencing of 3 samples only may help you to find error in digestion. If sequencing show heterozygote genotype of semidigested samples, it is not confirmation of your digestion protocol unless you perform sequencing on all samples.

Confirmation of your digestion is complete digestion of a sample that could be a known PCR product or plasmid

Use of plasmid is easy

You don’t need a specific plasmid, you can find a plasmid in your laboratory or your near laboratories. Every plasmid has a defined sequence that is available. You can check the sequence of plasmid for digestion (easily in webcutter 2 : http://rna.lundberg.gu.se/cutter2/ ) for digestion of HincII and size of digested fragments

Perform a digestion on plasmid and complete digestion of plasmid as expected pattern will confirm you digestion step.

You could check your friends PCR products for defined site of HincII and use as positive control too.

Regards

Hi, very interesting i used the same enzyme(HincII) to do RFLP recently,  i am sure imcomplete digestion happened since i used positive control. I think it's because the enzyme recognize blunt end and its effiency very low.  Could you please let me know how you solve the problem at last? 

Regards