Hello,
I need some help!
The aim was to detect the genotypes of various unknown samples with the H36D mutation using BCI I which abolishes the restriction site of a mutated individual.
Our expected fragment sizes were as follows:
Homozygous Wild-type: 161b & 247b
Heterozygous: 408b, 161b, 247b
Homozygous Mutant: 408b
In the image i have uploaded, the first and last well contain a faint upper band of expected size followed by the two smaller bands that are brighter. Does this mean the plasmid is uncut because it becomes darker therefore a Homozygous Wild-type genotype? Or is it a heterozygous as you can observe the three fragments of expected size?
Also in well 8 (not counting first two after MW marker) the first band is really bright but you can still see 2 really faint bands, would you call this a heterozygous genotype?
The MW marker 2-log DNA ladder.
Thank you in advance,
Alexia
Hi Alexia,
First of all, are these PCR products or plasmids. Going by size it seems they are PCR products (sizes matches the bp mentioned above). Considering this I have few suggestions
1) As the size of the two alleles is same with one polymorphism resulting in the restriction site, you'll have to be careful in distinguishing the heterozygotes and partial digestions.
2) The heterozygote will have both the alleles in almost similar amount whereas the partial digest will have a lower upper band. This can be verified by increasing the amount of restriction enzyme and time on incubation, The pattern of heterozygote will not change while the upper faint band (in suspected heterozygotes will completely disappear).
3) The amounts of products should be similar for such assays and there should not be inhibitory/interfering substances that affects restriction digestion
4) going by this I'll consider, only sample 4,5,7,12,15,19 and 24 as heterozygotes. 1-2 other suspected samples can be checked by allowing complete digestion.
5) Also see that the faint bands of other allele may also come due to sample cross contamination, if you are not careful.
I hope this helps..
hi Alexia,
I think Ajay has answered this very well and and would only emphasise some of the points he makes. I think the existence of the 2 small bands defines the normal state but that incomplete cutting may look like heterozygotes so the intensity of the upper band should be considered. It is larger so traps more ethidium bromide so should look brighter ( darker in negative display ). Also in the heterozygous state the heteroduplex generated from a normal/mutant strand association will not cut so it should look strong. Thus I think sample 3 is homozygous normal but is incompletely digested. Sometimes you need a little more enzyme with cutting pcr products as they are defined by cutting lambda dna which may have cut sites every few thousand bases but your pcr product will have a cut site every molecule possibly.. When you do have a heteroduplex sometimes the uncut heteroduplex runs very slightly slower than the uncut homoduplex of the same size which can be useful.
having said that I think that sample 8 could be a heterozygote badly cut but the bands are so weak that spillage from the samples either side is possible so it should be repeated.
I think sample 11 in the lower gel is homozygous cut and incomplete digestion.
think you could either cut for longer or use a little more enzyme and be very careful where a weak pair of bands/band is in between 2 strong samples or possibly leave a space between samples to minimise sample leakage
I think the last sample on the lower gel is homozygous cut but a partial digest and should be cut for longer because the upper band is so weak and it should be as strong or stronger than the cut band.
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