Southern Blotting Confusion

[betterfuture]

Advertisement - Members don't see this ad

Researchers conducted an experiment by comparing DNA samples from 3 individuals: homozygote for the normal ß globin gene, homozygote for mutant sickle cell allele and heterozygote for a carrier.

Each DNA sample is mixed with the same restriction enzyme, DdeI. Digestion leads to thousands of fragments. Gel electrophoresis is conducted forming a characteristic of bands and treated later with a stain. The gel is transferred and via capillary action the alkaline solution is pulled through the gel, denaturing the DNA in the process. The nitrocellulose blot is exposed to radioactive labeled probe. The probe is single stranded DNA complementary to ß globin gene. A sheet of photographic film is laid over blot and exposes image corresponding to those bands that base-paired with the probe.

Question
1)Why is it that the radioactive probe base paired with the mutant sickle cell DNA strand when it does not complement the sequence of a regular normal ß globin gene?

2)Restriction enzymes are of many kinds, so in this case the DdeI restriction enzyme did not cut through the gene sequence, it only made fragments that contained the intact sequence of ß globin gene? But in other cases, there are times when restriction enzymes cut through a specific gene sequence. I am just trying understand restriction enzymes as a whole because it kind of confuses me.

Thanks and I would appreciate the help!

 

[yeasureyoubetcha]

1)Why is it that the radioactive probe base paired with the mutant sickle cell DNA strand when it does not complement the sequence of a regular normal ß globin gene?

Even though the mutant sickle cell DNA strand doesn't share 100% complementary with the probe sequence, there's still enough for it to bind tightly. You might see a base skipped over or or a small hairpin form if there's a few mismatches

2)Restriction enzymes are of many kinds, so in this case the DdeI restriction enzyme did not cut through the gene sequence, it only made fragments that contained the intact sequence of ß globin gene? But in other cases, there are times when restriction enzymes cut through a specific gene sequence.

Yes. They will have different cut sites. In this example, the B-globin gene doesn't even need to be intact. Only the sequence that the probe binds to needs to be intact which is considerably shorter ~20-30nt.

 

[betterfuture]

Hey man! Thanks for your response! But for the second response, I am confused. How is it that the ß globin gene does not need to be intact? If the fluorescent probe is to complementary base pair with the gene and that gene is the ß globin gene, how is that possible? Just trying to get the whole idea. Thanks!

 

[yeasureyoubetcha]

How is it that the ß globin gene does not need to be intact? If the fluorescent probe is to complementary base pair with the gene and that gene is the ß globin gene, how is that possible?

Remember the Dde1 digest happens before the probe is added. The only time that the probe will not bind in this case is if there is a Dde1 cut site in the middle of the B-globin sequence that the probe binds to. Thus the probe would be unable to find its target sequence. I don't think anybody makes a radiolabeled probe that is the entire length of a gene, it would be expensive. I work with fluorescent probes at work and most vendors allow 18-40 nt long when ordering.

Also be careful not to mix up fluorescence with radiolabeling. They are used for different things.

 

[betterfuture]

Oh yeah forgot. It's radiolabeling. I was studying DNA technology and got mixed up with the two. Though I believe radiolabeling and flourescent hybridization do similar things: they complementary base pair with the sequence of a gene, right?

And for your response, are you saying that probes are not the whole complementary base sequence of the gene, say the ß globin gene? They only are a part of the sequence? How long ~30n.t long you said right?

 

[yeasureyoubetcha]

Oh yeah forgot. Its radiolabeling. I was studying DNA technology and got mixed up with the two. Though I believe radiolabeling and flourescent hybridization do similar things: they complementary base pair with the sequence of a gene, right?

Radiolabeling is good for seeing if a sequence is present as in the case you described as well as others I'm probably not thinking of. Fluorescence has numerous uses. qRT-PCR for quantitation. FISH probes can look for presence and subcellular localization of RNA or DNA. These are just few.

And for your response, are you saying that probes are not the whole complementary base sequence of the gene, say the ß globin gene? They only are a part of it the sequence?

Correct. But you can still get away with mostly complementary to a stretch of sequence and accomplish the same thing.

How long ~30n.t long you said right?

20-40nt is typical. This avoids probes dimerizing.

 

[betterfuture]

So how likely would it be then that another sequence of some other gene gets bound to the radiolabeled probe? Is that even possible? Because as you stated that the probe is not the whole gene sequence, only part of it~30n.t. long.

 

[yeasureyoubetcha]

So how likely would it be then that another sequence of some other gene gets bound to the radiolabeled probe? Is that even possible? Because as you stated that the probe is not the whole gene sequence, only part of it~30n.t. long.

It could happen. Crapshoot. Is it likely? Maybe, maybe not. Do out the probability. It could bind sequence with high homology. With my work, we target a consensus sequence for a repeat element in the genome that has some sequence variability among elements and still get the job done.

 

[betterfuture]

Also, since you work in the lab, I am guessing, from what you said above, could you also explain dideoxy chain termination for gene sequencing. As in I understand didNTP's are used to terminate the DNA replication but they - the researchers- add them at random so that different size strands are made. But if they do this at random how do they make sure the sequencing of every base is complete. Do you understand what I mean?

 

[yeasureyoubetcha]

Also, since you work in the lab, I am guessing, from what you said above, could you also explain dideoxy chain termination for gene sequencing. As in I understand didNTP's are used to terminate the DNA replication but they - the researchers- add them at random so that different size strands are made.

I've never done it. Don't know anyone who has.

I'll venture a guess. They may create separate terminating reactions for different ddNTP concentrations, then pool the rxns for each ddNTP. After they're done, they go on the gel. One lane per pooled ddNTP--> For this, definitely make sure to read from the bottom of the gel haha

But if they do this at random how do they make sure the sequencing of every base is complete. Do you understand what I mean?

The probability that the terminating NTP will get incorporated is dependent on the concentration of said ddNTP. Also, if you're missing a band on a sequencing gel thats a pretty big tell.

 

[betterfuture]

I'm sorry. Can you rephrase what you just said. That whole paragraph just went over my head. Sorry, for that. I have little research experience so I find it a little harder to understand thoroughly the procedures.

 

[yeasureyoubetcha]

This is what I think is done:
1.) Create individual tubes with multiple concentrations for each ddNTP (say 5 concentrations each for ddA, ddT, ddG, ddC)
2.) Run PCR
3.) Pool the reaction products for 5 concentrations of ddNTP for each ddNTP. (Should have 4 pools at this point)
4.) Run the pools out on a gel
5.) Read from the bottom up

 

[betterfuture]

What does a "pool" mean?

 

[yeasureyoubetcha]

What does a "pool" mean?

Combine. Like you and your friends would pool your $ for a sports bracket.

 

[betterfuture]

Were you describing the dideoxy chain termination, cause if you were, I am now more confused.

The fragment of DNA strand to be sequenced is denatured. The ingredients, a primer, DNA Polymerase, dNTP and ddNTP(without the OH and are fluorescently tagged) are used. From there on out, it just confuses me cause they add at 'random'. And that's where I got confused as to why they do it at random when they can time the reaction. Right? The polyacrylamide gel part I have down because I know the shorter strands will be at the bottom.

 

[yeasureyoubetcha]

The fragment of DNA strand to be sequenced is denatured. The ingredients, a primer, DNA Polymerase, dNTP and ddNTP(without the OH and are fluorescently tagged) are used.

I don't think its fluorescently tagged. I think you can just use a regular old DNA stain for this.

From there on out, it just confuses me cause they add at 'random'. And that's where I got confused as to why they do it at random

They "the experimenter" aren't doing anything at random. Taq will add the nucleotides at random (dNTP or ddNTP) within each reaction. The probability that it adds a ddNTP will depend on the concentration of that ddNTP. The concentations of ddNTP the experimenter uses may be arbitrarily chosen or may have been optimized through previous experiments.

when they can time the reaction.

I'm not exactly sure what you mean by this. PCR isn't done by time, its done by number of cycles. 40 cycles is typical. Sequencing may be less

 

[betterfuture]

I think the procedure you described is a little different from what I have understood. The method I describe does not incorporate PCR anywhere.

 

[betterfuture]

And this is coming straight out of a book. The procedure.

 

[yeasureyoubetcha]

Yea you're right. I don't know why I was thinking that. Just ignore the bit about he PCR step

Sent from my iPhone using SDN mobile

 

[betterfuture]

Thanks for the help though!