PCR and Southern blotting differ per methodology and the majority of us here know these techniques in detail. But I was wondering why a researcher would prefer to conduct a Southern Blot over PCR, as both methods seek to identify a sequence(s) of DNA/RNA and as PCR has provides more advantages to offer?
Some (but certainly not all) applications of southern blot that you cant easy do with PCR,
1. Detecting multiple homologous genes in a genome - PCR tends to be very specific and when two bands are seen, it is more likely non-specific or contamination
2. Detecting orthologous or paralogous genes in similar or distant species where you might not know anything about the sequence divergence and hence primer sites (you can increase or decrease specificity depending on hybridisation conditions) - if you dont know the primer sites, you cant do this
3. Detecting insertions of a plasmid/viral vector when making stable transgenics and determining copy number (similar to #1 really). Cant with normal PCR, qPCR you could
4. Easier to multiplex / detect multiple products
5. Similar to Kyle - you can analyse methylation patterns that affect restriction sites (as you digest with a restriction enzyme before probing)
6. Agree with Christian - replica plating - much easier/more time efficient than PCR
There is a couple of the top of my head. I am sure there are lots more out there.
If you're simply identifying the presence of a certain DNA sequence, I think that both methods are valid. We're currently using Northern blots to detect post-transcriptional RNA modifications (such as formylation), and I imagine that a Southern blot could be useful in a similar setting: to separate DNA based on modifications rather than nucelotide sequences.
Thanks Mr. Kyle Strickland for your answer.
But I still had confusion, as modifications are only post-transcriptional (i.e. in mRNA) and post-translational (i.e. in protein). Can you describe me DNA modifications ?
Southern blots are for example useful if you want to screen large clonal libraries. Here you screen more colonies faster than with PCR.
Mr. Christian, if you are telling about microbial colonies, then colony PCR is a fast and efficient way to screen DNA sequence directly from the colony.
Kindly elaborate your statement !
When you do PCR, you just see the amplified region. Maybe the gene you examine is broken right after the reverse primer. In this case, you might set another primer set partially overlapping the first amplified region . But, even if you get proper sizes of fragments by both PCR, you can not deny that each fragment comes from different regions--different chromosomes or 2 partial fragments in the genome. In addition, even if you have a band at the proper size, you never know it has a proper sequence before you validate it. It should be very rare, but what if a nonspecific band comes at the same size of your target. When you do lots of various different sample, one of them may be what you do not want. Technically, PCR gives various possibilities more than southern blot.
PCR used to be thought to be not good enough for quantification. Even if one tenth of cells have your target DNA in the genome , you can amplify the fragment. Even, today, you have to set up quantification methods carefully, if you have to avoid this kind of mis-interpretation. This kind of misinterpretation can happen by southern blot but you can easily figure it out if only one tenth of cell has your target.
@Muhammad: I am talking about screening genomic DNA libraries to find the one clone, which contains your sequence. Believe me, you don't want to do PCR on 10.000 clones. So you plate them on a few plates, make replica plates for later usage and then screen the plates for positive colonies. Relatively fast and straight-forward.
Some (but certainly not all) applications of southern blot that you cant easy do with PCR,
1. Detecting multiple homologous genes in a genome - PCR tends to be very specific and when two bands are seen, it is more likely non-specific or contamination
2. Detecting orthologous or paralogous genes in similar or distant species where you might not know anything about the sequence divergence and hence primer sites (you can increase or decrease specificity depending on hybridisation conditions) - if you dont know the primer sites, you cant do this
3. Detecting insertions of a plasmid/viral vector when making stable transgenics and determining copy number (similar to #1 really). Cant with normal PCR, qPCR you could
4. Easier to multiplex / detect multiple products
5. Similar to Kyle - you can analyse methylation patterns that affect restriction sites (as you digest with a restriction enzyme before probing)
6. Agree with Christian - replica plating - much easier/more time efficient than PCR
There is a couple of the top of my head. I am sure there are lots more out there.
You cannot classify DNA modifications as post-transcriptional or post-translational for obvious reasons. I was thinking along the lines of chemical modifications, such as methylation or formylation.
Thank you all RGS for your answers.
It really helped clearing my concepts.
Southern blots can be very useful if you are working with a species without a fully annotated genome sequence available. Unlike PCR-based approaches they will give you additional information about closely related isogenes (if you chose your hybridization probe wisely). When you want to determine the number of T-DNA insertions into a plant genome, then Southern Blots are also great as they give you additional information about the size of the T-DNA insertion and the number of independent insertions versus multiple insertions into one locus.
You can establish the molecular weight of the target sequence, something that you cannot do in PCR.
Depends if you are talking old PCR. Results will be much better with qPCR, and will be less time consuming.
If the template is palindromic, you could have problems during the annealing step of PCR as the ssDNA template could form hairpins.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques