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problem of restriction enzyme digestion


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3 replies to this topic

#1 jamestoon1

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Posted 20 June 2012 - 07:09 PM

hi all,

i am doing SNP genotyping.
my pcr product is 280 bp
if the subject is homozygous wildtype, the RE will not cut, so i will get a single 280 bp band

if the subject is homozygous variant, the RE will cut, and i will get a 240 bp band and a 40 bp band
if the subject is heterozygote, i will get all 280 bp, 240 bp and 40 bp bands

now i have one problem
i cannot be sure whether the RE cuts or not
for some samples, the 240 bp band seems to be present but the band is very very faint (indicated by the red arrows in the following gel)
sssa.JPG

so, my question is, how should i interpret these? are they heterozygote or homozygous wildtype?
what can i do to improve RE cutting?
the protocol mention use 1 unit of enzyme and incubate for 1 hour. i have already tried increase the enzyme to 10 units and incubation for up to 6 hours, but the appearance of the bands are still unclear.
i have several thousands of samples to run, so it would be infeasible to send all for sequencing.

#2 jamestoon1

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Posted 21 June 2012 - 09:23 AM

can anyone help me??

#3 jamestoon1

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Posted 21 June 2012 - 07:18 PM

bump

#4 phage434

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Posted 22 June 2012 - 03:57 AM

It's very hard to give you advice. You definitely need some good and reliable positive controls for each of the conditions you are trying to detect. I sense that the RE digestions are not going to completion, which makes the analysis quite difficult. In principle, if you see any of the shorter bands, you have the cut construct, but you need to differentiate that from the construct in which all of the product can be cut. This means you need complete digestion, which is quite difficult to achieve, and very difficult to achieve in an uncontrolled environment. Controls for the efficiency of your cutting will be absolutely essential. Perhaps there is a diagnostic you can use that will detect the other construct with similar digestion with a different enzyme. That would be much much better than relying on total digestion as you means of differentiating strains.




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