i think you already have the answer Ariza. If the snp/mutation creates the restriction site then a single uncut band of 480 is homozygous normal. The existance of 3 bands means one uncut ( normal ) band and one cut band from the snp and is heterozygous and the pattern of no band at 480 but only 376 and 104 is a homozygous snp. Sometimes in this situation in the heterozygous state the abundance (intensity) of the uncut band is disproportionally high as pcr creates both n-n and the similar n-m heteroduplex both of which do not cut with restriction enzymes but the number of bands is definative

i think you already have the answer Ariza. If the snp/mutation creates the restriction site then a single uncut band of 480 is homozygous normal. The existance of 3 bands means one uncut ( normal ) band and one cut band from the snp and is heterozygous and the pattern of no band at 480 but only 376 and 104 is a homozygous snp. Sometimes in this situation in the heterozygous state the abundance (intensity) of the uncut band is disproportionally high as pcr creates both n-n and the similar n-m heteroduplex both of which do not cut with restriction enzymes but the number of bands is definative

One potential problem could be different mutations at the site, that do not create RFLP.

So one could only say that the rest 24 samples without RFLP do not have this particular mutation. Ariza, it will be prudent to check frequency of all possible mutations at your site in the SNP database.

I think you have clear cut results on your gels answering your questions. Your samples showing only one band on the gel are homozygous and both alleles have not been digested with restrictions enzyme. The samples with the bands are heterozygous, means one allele has no restriction site (450bp bands) and therefore remained undigested. Whereas the second allele has the restriction site and has been digested with restriction enzyme and resulted into two fragments (376bp and 104bp fragments). In conclusion RFLP results are conclusive on gels and you don't have to sequence to know your answer. I hope it will help you.

thanks a lot everyone for answering my question.

but, one thing I don't know is;

why sample number 1 has different thickness of 480bp and 376bp if it compared with the other heterozygous sample. I mean like this, the sample number 1 has the thick band on 376bp, the 480bp and 104bp is thin (like on pictures i attached). but the other samples had the thick band on 480bp, the 376bp and 104bp is thin (like on pictures i attached). so, can anyone explain to me? if there's a journal that could explain this question, please give me the access of the journal.

or did I do some mistakes when RFLP?

thks in advance.

hi,

some thoughts.

in figure 2 the small band is weak because it is small and traps ethidium bromide weakly so the same amount of 376 band is stronger because it traps more etbr. the 480 band is very much stronger for a different reason. When the sample is a heterozygote the last cycle of pcr creates single strands of normal and mutant dna which re anneal to each other creating n-n , m-m and a lot of n-m . both hte n-n and n-m heteroduplex do not cut  with restriction enzymes so the is more full length uncut dna present.

I think sample 1 is homozygote mutant  but a partial digest. Remember the unit of enzyme are calculated by cut sites in large dna molecules or genomic dna where the average cut sites are many kb apart. In your pcr product there may be one or 2 cut sites every 480 bases so you need to use a little more enzyme and cut for longer and have a control homozygote mutant as a control in this experiment.

A less likely explanation is that you have either some pcr contamination ( the negative control sample will tell you this) with normal product or some of the cells that you are analysing in each sample are chimaeric so that you have normal and mutant cells in the same sample

well, thankyou for the answer. I'd like to discuss this phenomenon with you, sir..

once again, I did RFLP again and this time I got the non SNP mutation and Sample number 1 still included (like on the picture i attached).

this time, I got the different thickness of every bands (480bp, 376bp and 104bp). the 480bp band is thick this time, but the 376bp is thinner and the 104bp is almost invisible, at least, when I looked it by Transiluminator, the 104bp is still visible but very thin and my camera capture it (when I negative the photo, it looked like so thin like when it was on transiluminator). how could it be possible? is it still associated with the n-n, n-m, m-m when I do the PCR?

if it is, how can I not to cut the heteroduplex?, and am I should designing my primer again?

thks in advance, sir..

Hi Ariza, Paul's thoughts above, explaining different bands thickness was right on the mark. When you do high cycles PCR and have related but slightly different sequences, you will have this phenomena forming different heteroduplexes. To avoid this, one could do so called rescue PCR: after finishing you initial PCR, dilute it with 8x excess of original PCR mix (without adding genomic DNA) and do just 2 cycle PCR. Most of your products will be homoduplexses now and your cutting pattern will be closer to expected. About your sample #1, it is hard to explain such a different patterns in two different cases. Redo sample 1 with rescue PCR and see which pattern is reproducible.  

hi Ariza,

Dmitry is right with his solution  using rescue pcr which should help. I would expect your result with the strong 480 band comprising all of the n-n .n-m and m-n being most of the product. It is important mathematically to consider n-m as distinct from m-n. You cannot cut a heteroduplex because type 2 restriction enzymes need a palindromic recognition site ( which is often also the cut site) so in a heteroduplex wher eperhaps a C sits opposite an A there can be no recognition of the sequence therefore no cutting. What does worry me is that you do not include a no sample blank in your experiments...if you have any pcr contamination of normal product the eventually every one of your samples will appear to be either normal or heterozygous and the uncut band will get stronger as well as being present when it should not be.. You must run both a positive (het sample) and a zero sample blank in every experiment to be sure of your results. There is no need to redesign primers unless you do get bad contamination problems but i would run my gels at a lower voltage for a longer time as your size standard seems to have run far from the wells. Sometimes small band do get faint if you keep them on the transilluminator for too long as uv light breaks up your dna so that it traps less ethidium bromide so take photos quickly

well, I will do the rescue PCR then, sir. I'll discuss it with my lecturer.

but, what what is zero sample blank? is it a control? and what do you suggest for the zero sample blank? or is it just a blank space when I do the electrophoresis?

I'm sorry, for that question. I'm a beginner so I don't know much things about this, and still learning.

sorry for questioning a lot..

Another good method for SNP genotyping is Small Amplicon Genotyping method. Here is the link;

http://biofiredefense.com/pdfs/LightScanner/SmallAmpliconGenotyping-AppNote-0124.pdf

I had already experienced about it and it worked well. It is also high throughput, high sensitivity and cost effective method.

Good luck.

thks sir for the another method, I'll discuss it with my lecturer.

you need to run a sample in every pcr amplification set which deliberately has no dna sample in it. PCR is so powerful ( in 10 cycles you have 1000 times more product than you started with )  that the tiniest amount of previously amplified product transferred from the tubes or from the electrophoresis buffer on your hands or pipettes may get into a reagent by accident. then every tube is contaminated and you will get great amplification of the contamination product in all of your tubes which will confuse the real answer from your samples. I use water instead of a dna sample. Some people suggest that the water is left open on the bench to detect contamination in the working area but  it must be  something which should not amplify so the water that you make your reagents in is good. The control must be treated exactly as your other samples and should give no amplified band. If it shows a band then you have a serious problem but one that can be dealt with

well, thks for all these answers and suggestions, Mr. Paul Rutland. I'll try my best next time. thankyou so much sir.

good luck Ariza and if you have any further questions on this project you can always email me if you think I can help

paul

yes, sir.. thankyou so much for your kindness and your helps.

Ariza