[rleung]

Hey,

I am confused about the ribonuclease mismatch cleavage as a means of detecting single nucleotide polymorphisms.  In my book, it says that this is a method of scanning the DNA strand for a mismatch, but I don't see how.  If you are separating the DNA strands and then using the DNA as a template for the synthesis of a complementary RNA strand, then how would you be able to detect a mismatch?  Wouldn't the mismatched deoxyribonucleotide automatically be paired up with the correct ribonucleotide?

Thanks.

[Yggdrasil]

What organism does this occur in and what enzyme specifically is doing the cleavage?  In prokaryotes, DNA is methylated some time after replication.  So, in DNA mismatch repair, the newly synthesized strand will not be methylated and can be distinguished from the template strand (which has methylations).

[rleung]

The enzyme is ribonuclease A.  But the book is using this method as a way to determine SNPs, and I don't see how this works.  Let's say there was a SNP on the parent (methylated) strand.  If we synthesize an RNA strand complementary to the parent strand that is unmethylated, it will just place the correct nucleotide to base pair with the SNP site.  So, I guess I don't understand how this can allow one to determine that there is a SNP.

[Yggdrasil]

Is this a natural way of detecting mismatches or an assay used in labs?  I don't think RNase A is used in any natural DNA repair pathways.  Anyway, RNase A just cleaves single stranded RNA.  How is the complementary RNA strand made?  Perhaps it would help if you posted a summary of the protocol for using RNase A to detect SNPs.

[rleung]

This is an assay used in labs.  Hmm...well, it doesn't go into much depth about it.  It doesn't explain a protocol.  It talks about both the ribonuclease mismatch cleavage, the chemical cleavage method, and enzyme cleavage method (all cleavage-based methods).  Here is the summary:

"Several techniques exploit the fact that mismatched bases are sensitive to binding or cleavage by enzymes or chemicals.  After PCR amplification, the wild-type and variant products are subjected to denaturation/renaturation to create heteroduplex molecules.  After incubation with resolvases or chemicals, the products are resolved electrophoretically side by side to score for the presence of mismatch-cleaved DNAs.  Cleavage methods mostly work on heteroduplex molecules, cleaving the helix at the distorted region caused by the mismatch.  This means that homozygous or hemizygous mutations would not be detected unless the sample is hybridized with a reference sample to form the mismatched heteroduplex.  These methods are able to scan larger fragments than conformation-based techniques, and the size of the cleaved product roughly indicates the localization of the mutation; however, they require considerable post-PCR manipulation."

It goes on to explain the different protocols:

"Ribonuclease mismatch cleavage: the single-strand specificity of RNase has been utilized to digest RNA:RNA or RNA:DNA heteroduplexes.  The technique has been adapted to commercially available nonisotopic RNase cleavage assays (NIRCA) with higher mutation rates than in the original versions.  However, the major drawback of these methods is that they require in vitro synthesis of RNA."

I just don't get how this could lead to detection of an SNP in vitro.  How would ribonuclease know that a certain base is incorrect?

Ryan

[Yggdrasil]

Ok, here's how I see the procedure working.

You take a reference DNA (or RNA) strand and then mix with RNA containing the sequence you want to test.  The reference DNA should be complementary to your RNA and hybridize to the RNA.  Wherever there is a mismatch (due to an SNP), however, the bases will not pair and the helix at that position will be distorted.  Distortions in the DNA-RNA double helix can be recognized by RNase A, which has the ability to cleave single stranded RNA, but not RNA hybridized to DNA.  So, while RNase A will not be able to cleave most of the RNA, it will be able to cleave at any positions where the RNA base is not correctly paired with a DNA base.  So, the presence of cleaved RNA after treatment with RNase A indicates that the RNA did not hybridize exactly with the DNA, indicating that the RNA sequence differs from the DNA sequence by at least one nucleotide position.

[rleung]

Hmm, so what you're saying is that the SNP will cause conformational changes in the SINGLE strand of DNA that will prevent RNA from base pairing with it?  I knew that base mismatches usu. cause conformational changes, but I thought that that existed in duplex form.  In this case, the conformational changes would carry over to the single-stranded form, right?

[Yggdrasil]

No.  In the DNA-RNA hybrid, there will be a "kink" where the bases of the DNA and RNA cannot base pair.  The local conformation of the RNA around this kink is similar enough to single stranded RNA that RNase A can recognize and bind to the RNA and cleave it.  Base mismatches will not cause conformational changes that carry over to the single stranded form.

[rleung]

Hmmm.....I am still confused.  Here is what I "assume" we are trying to do:

There is an SNP in a cell.  To determine it, we isolate a segment of DNA from the cell, and we separate the strands.  How does hybridizing RNA that is synthesized in vitro help to determine the presence of the original SNP that existed within the cell???  According to my understanding, we are trying to investigate the original SNP in the cell, not any mismatch base pairing that occurs when RNA hybridizes to DNA that originates from the cell.  Your last post makes it sound like we are just trying to detect where a mismatch occurs in a DNA-RNA hybrid, but aren't we, instead, trying to find a base mismatch in the original DNA-DNA duplex in the original cell?

[Yggdrasil]

A single nucleotide polymorphism is a difference in sequence between two individuals in a population, not between two strands of a DNA double helix.

http://en.wikipedia.org/wiki/Single_nucleotide_polymorphism

So what you would do is take a reference DNA strand, either isolated from an individual or chemically synthesized and take a RNA synthesized from another individual's DNA.  Hybridize the two, treat with RNase A, then visualize the fragments on a gel.

[rleung]

Thanks.

Wait, so what you're saying is that the complementary strand of RNA that is complementary to the target DNA under investigation is not synthesized from ribonucleoside triphosphates?  I thought it was synthesized from the individual nucleotide monomers (much like the transcription process).  So, are you saying that, instead, you take an already-synthesized segment of RNA isolated from another individual and hybridize it with the target DNA? 

[Yggdrasil]

You take some DNA isolated from the individual whom you want to test for the presence or absence of the SNP.  Then you somehow in vitro transcribe RNA from that segment of DNA and use this RNA for the assay.  I'm not sure how they do this since most SNPs of interest are in non-coding regions, regions which are not transcribed and have no promoter to recruit RNA polymerase.  Maybe this assay is useful only for SNPs in transcribed regions of the genome. 

[rleung]

Hmm, I know I must sound so stupid, but I still can't understand how this allows one to detect for the presence of an SNP on the original strand of DNA that was present in the cell.  Here is how I understand it:

1) wild-type DNA:

AGTC
TCAG

mutant SNP-containing DNA:
AGGC
TCCG

There is an SNP in the 3rd nucleotide.  If you separate the strands and isolate one, you may get the following strand:

AGGC

Then you take RNA synthesized in vitro to make the complementary strand, so you now get..

AGGC
UCCG

I don't see how the synthesis of the complementary RNA would allow for detection of the SNP in position 3.  If you separate the original DNA strands, how would RNA know that position 3 is an SNP from the more common wild-type DNA, unless my understanding of SNP's is incorrect somehow...

[Yggdrasil]

Wild type DNA (reference sample)
5' - AGTC - 3'
3' - TCAG - 5'

Test DNA (contains SNP)
5' - AGGC - 3'
3' - TCCG - 5'

Transcribe one strand of the test DNA into RNA:

5' - CGGU - 3'

Hybridize the test RNA with reference DNA:

5' - AGTC - 3' (DNA)
      | |   |
3' - UGGC - 3' (RNA)

RNase A will be able to cleave where the mismatch occurs.

I googled "RNase A, SNP" and found the following review article describing RNase based assays for SNP detection.  I haven't read it yet, but it looks like it might be useful to you:

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11524730&dopt=Abstract