Visualization of DNA:RNA hybrid on TBE-UREA GEL - (Mar/04/2015 )

Hi everyone,

I run 10% TBE-UREA gel to visualize DNA:RNA hybrid and ssDNA (single stranded DNA). Goal of the experiemnt was to see enzyme digestion effect of enzymes S1 nuclease and Mung bean nuclease on DNA:RNA hybrid and ssDNA. 10 bp DNA ladder was used. Urea-buffer was added to all the samples and the ladder and was heated to 70 C for 3 mins before laoding on the gel.

PFA attached pictures of the run.

There are two bands in the first picture i wanted to know which band is DNA and which band is RNA? As both them are single stranded and same number of nucleotides which is 48 then why they form different bands ?

IF they do form different bands is the lower one DNA becasue RNA has higher mass to charge ratio and will travel slower.

The second gel is just ssDNA with and without enzyme treatment. Both gels were run simultaneously on life technologies mini gel tank. Bands look similar to RNA band in gel one but the size coressponds to DNA if you go by my reasoning !!

Can anyone help out ... i can give more details if that helps but i think i have mentioned all the information.

single stranded dna is ~16 Da/base less massive than rna so one would expect the rna strand of the hybrid to be more massive than the dna strand.

is your ssdna the same size as the dna strand of the hybrid? if so then the ssdna identifies which band is dna.

The dna strand of hybrid and ssDNA are both of length 48 nucleiotides. Before this i had done experiemnts with DNA:DNA and ssDNA digestion and ran on urea gel. Then also the denatured dDNA and ssDNA which is the same concentration and amount had different bands. Denatured dDNA would be about 10BP higher than ssDNA on the gel.

WHy so ?

The band should be at the same position and same intensity logically but it is not !! any valid reasons. i did heat the samples with urea buffer so i think they were all denatured...

Any suggestions as to what is happening here..

if the dna strands don't have the same amount of each nucleotide then they may have different masses.

heating decomposes urea. the strand could have a hairpin that reduces it's 3 dimensional profile and allows it to run faster.

Both ssDNA and the DNA of the hybrid are of the same length.

same length is okay but are they identical in numbers of each nucleotide?

yes

maybe the decomposed urea has modified the strands (it will carbamylate proteins, not sure what it would do to nucleic acids).

how reproducible is the difference in size?