What if this is an XYY situation ( complete or mosaic) either as total chromosomes or a translocation of the sry region to an autosome inherited with the expected Y. AS this is a pcr technique are you sure that your negative pcr control ( no template) is fully negative and you can be sure that there is no contamination.. I assume this is a human sample and that there is no pseudogene for sry. Has chromosome spreads been run or Y chromosome polymorphic markers in the sry region?.

It seems that in 80% of XX males there is a translocation of sry onto the X so translocations are not excessively rare in this region

Paul Rutland is right, as always, but you should also test your PCR in silico to see if you're amplifying the right sequences (http://genome.ucsc.edu/cgi-bin/hgPcr).

all the best

fred

Human samples?

Cattle samples....

Paul Rutland

Thanks for ur insights..

1. Im sure there is no contamination and Non template control is blank and perfect

2 . Im working on cattle samples...

3. SRY is reported to be single copy in cattle... and i doubt SRY is polymorphic in cattle.... needed to be checked however THREE GENOTYPES OF SNP is not expected...help me out how my discussion should be focused..

Thanks in advance

What are your 3 genotypes? Are they all sequenced or are these different sized fragments generated by one enzyme. If this is the case what sizes are these genotype bands and are they all of roughly the same intensity? What size is the uncut amplimer. A picture would be good to see please

Is any one band looking larger than it is supposed to be compared to the uncut control?

Is the enzyme that you are using BfA1 Jeevan C.

A simpler explanation to those given above would be that the enzyme that you are using cuts twice within your amplimer and both of these sites are polymorphic leading to three different cut patterns depending on whether each site cuts or does not cut

I deeply apologize for the late reply sir,

> the SNP of interest is SRY 641G>T of Bos taurus

> the enzyme used is MseI

>sorry the organised pic is not captured but ill attach the crude one (image attached)

> the products obtained are as follows:

GG= 542+201

GT=542+201+124+77+(undigested or actual length polymorphism: not sure of amplicon length 666; present only in heterozygote)

TT=542+124+77

> I think you are right Paul Rutland , may be improper digestion by RE coz apart from GG and TT fragments I'm getting all different lengths possible by improper digestion for example 659 product length(743-77=666)

so I feel GG is GG, TT is TT, GT is actual TT. am I right?

thank you so much

Can you send me teh sequence of the amplimer between the primers please Jeevan C.

thank you

paul

F: CGC AGT GCA GTC GTA TGC TTC TGC

R: AGC GCC TTT GTT AGC GAG AGT AAG G

primers

Thank you for the primer sequences . Jeevan C.

I have blasted them and my comments refer to the sequence

cagtgca gtcgtatgct tctgctatgt tcagagtatt

14281 gaacgacgat gtttacagtc cagctgtggt acagcaacaa actactctcg cttttaggaa

14341 agactcttcc ttgtgcacag acagtcatag cgcaaatgat cagtgtgaaa ggggagaaca

14401 tgttagggag agcagccagg accacgtcaa gcgacccatg aacgccttca ttgtgtggtc

14461 tcgtgaacga agacgaaagg tggctctaga gaatcccaaa atgaaaaact cagacatcag

14521 caagcagctg ggatatgagt ggaaaaggct tacagatgct gaaaagcgcc cattctttga

14581 ggaggcacag agactactag ccatacaccg agacaaatac ccgggctata aatatcgacc

14641 tcgtcggaga gccaagaggc cacagaaatc gcttcctgca gactcttcaa tactatgcaa

14701 cccgatgcat gtagagacat tgcacccctt cacatacagg gatggttgtg ccaagaccac

14761 atactcacaa atggaaagcc aattaa gccg gtcacagtcc gtgatcataa ccaattcact

14821 cctgcaaaag gagcatcaca gcagctggac aagcctgggc cacaataagg taacattggc

14881 tacacggatt tcggcggact ttccct gtaa caaaagctta gagcctggac tttcttgtgc

14941 ttattttcaa tattgactt c cttactctcg ctaacaaagg cgct

Which probably varies slightly from your amplimer but I will use it as an example and you can compare it to your sequence. The MSe1 site at 14786 will cut in all samples giving sizes of 540 and 201 and in partial digests of 741. Partial digests are a problem with pcr product as the partial looks just like an uncut sample and pcr product may have a higher incidence of cut site than genomic dna so we have to cut for longer and use more enzyme than usual. Your gel is excellent but it really should have included a couple of deliberately uncut samples for comparison purposes.

The 201 base fragment has the motif TTAG at about 14921 which can be mutated from TTAG to TTAA and then this also cuts with MSe1 to give fragments of 135 and 66.as is seen in your gel samples top3 and bottom 14. The remaining samples with 2 small bands must be cut again for longer and with more enzyme ( overnight) Normally I would expect the 201 bans to be as intense as the 2 smaller bands combines . I agree that 201 looks weaker and is probably a partial digest but you cannot be absolutely sure so cut again and be sure.

The very larges bands are interesting. I think that what you have here is a heterozygote situation. So the pcr amplified strong bands so the last cycle of pcr melts the product and as there is a lot of product we get annealing not only of primers but reannealing of the 4 dna strands . Calling these strands N and M for the changes base we get NN.NM,MN and MM strands reannealing in the heterozygote situation. NM and MN have annealed with a bubble in the dna at the enzyme cut site so the enzyme cannot cut as the dna is not double stranded at that point. Also because of the bubble in the dna the dna runs slower ( apparently larger) through agarose gel. As You say this only occurs with heterozygotes. You may think that this is a nuisance but it is also indicative of the heterozygote existing so is diagnostic of a heterozygote. An uncut control sample of a heterozygote amplimer would show this clearly.

Thank you so much Paul Rutland for your detailed explanation in this regard. I understood the concept now, and I follow your suggestions. once again thank you so much for your time.

Thank you Jeevan C. If you do run these sample again try running the gel at lower voltage and a shorter time. The larger bands are not important and the smaller bands will be sharper and better defined. You can always do a short run, take a photo and then run further if you want overall separation and take 2 photos. It may make the gel clearer to read

Sure sir... I'll do... Many many thanks to you....🥰