Speaking of Qbank Exploitation...

[BlackNDecker]

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It appears there's a theme with Qbank Molecular Biology Q's that PCR is the test of choice.

Example:
A question dealing with CF asks which test would determine if the parents of a child with CF are carriers of the mutation?

A. Northern Blot
B. Southern Blot
C. PCR & sequencing
D. Western Blot

Kaplan says the answer is C.
I picked B. Kaplan says Southern blot is used to study DNA on a gross level (whatever that means 🙄 ) and could NOT be used to detect a single base pair deletion.
Loss of Phe is the result of a 3 base pair!! deletion (not a single base deletion as suggested in the explanation provided)...and Southern blot is the test of choice for prenatal Dx of Sickle Cell which is a 3 base pair substitution!

Can anyone help me out on this 😕

 

[PhillyGuy]

PCR is the method of choice if you have the sequence of the gene and if there a relatively small number of alleles that cause the disease so that you can sequence all of them to look for mutations. Southern blot is used to look for DNA fragments, not genes. It's common used to detect RFLP, such as paternity testing.

 

[BlackNDecker]

PCR is the method of choice if you have the sequence of the gene and if there a relatively small number of alleles that cause the disease so that you can sequence all of them to look for mutations. Southern blot is used to look for DNA fragments, not genes. It's common used to detect RFLP, such as paternity testing.

Thanks... 👍

Unfortunately, I'm still confused why you'd use Southern blotting to analyze RFLPs generated by the point mutations of sickle cell and phenylketonuria but you wouldn't use the same method to analyze cystic fibrosis due to a frameshift mutation?

HY Molecular Biology lists Cystic Fibrosis under diseases indicated for prenatal testing with DNA analysis under Southern blotting (p. 22 & 23 for those that have the book.)

For the sake of learning and not simply memorizing....If a researcher knew the sequence of any protein (CFTR in this example) and thus the DNA sequence... couldn't he/she use a restriction enzyme to generate fragments of the DNA --> then use Southern blotting to analyze the fragments generated? It seems like sequencing the DNA would be "reinventing the wheel" each time you wanted to diagnose a certain disorder. Furthermore, isn't PCR required to amplify DNA for any diagnostic technique that uses DNA?

 

[funkless]

For the sake of learning and not simply memorizing....If a researcher knew the sequence of any protein (CFTR in this example) and thus the DNA sequence... couldn't he/she use a restriction enzyme to generate fragments of the DNA --> then use Southern blotting to analyze the fragments generated? It seems like sequencing the DNA would be "reinventing the wheel" each time you wanted to diagnose a certain disorder. Furthermore, isn't PCR required to amplify DNA for any diagnostic technique that uses DNA?

I thought that was a shady question, too, but Robbins corroborates. Oh, well.

 

[PhillyGuy]

For the sake of learning and not simply memorizing....If a researcher knew the sequence of any protein (CFTR in this example) and thus the DNA sequence... couldn't he/she use a restriction enzyme to generate fragments of the DNA --> then use Southern blotting to analyze the fragments generated? It seems like sequencing the DNA would be "reinventing the wheel" each time you wanted to diagnose a certain disorder. Furthermore, isn't PCR required to amplify DNA for any diagnostic technique that uses DNA?

No. Unless the mutation is at the place where an endonuclease is going to cut the DNA, and thus generating the fragment, a point mutation will not alter the length of any of the resultant fragments. Therefore, you will not detect a difference. Therefore, Southern blotting is really not that useful for detecting small changes in the DNA.

On the other hand, if you know the sequence of the mutation, you can look for it with PCR. You don't have to sequence the entire gene, just the region around the mutation. It is only helpful for diseases like CF, where a single mutation causes the disease. It's less useful when you have allelic or locus heterogenity. In patients with thalasemia, for example, you don't use a PCR because too many mutations can cause the disease. So you use electrophoresis instead.

 

[BlackNDecker]

No. Unless the mutation is at the place where an endonuclease is going to cut the DNA, and thus generating the fragment, a point mutation will not alter the length of any of the resultant fragments. Therefore, you will not detect a difference. Therefore, Southern blotting is really not that useful for detecting small changes in the DNA.

So basically, only pick Southern Blotting for paternity disputes (i.e. RFLPs), correct? Is S. blotting still the technique of choice in forensics (i.e. VNTRs)?

So really, the question was asking "when do you sequence DNA?" The answer being, "when you have a Dz involving a point mutation or 3-base pair substitution that does NOT affect a restriction site." They could get really picky if they expected you to know which diseases affected restriction sites 😱

I got hammered on this question because it expected you to know that CF is caused by hundreds of different 3-base pair substitutions...I ASSumed all cases were caused by Phe508 substitution.

 

[PhillyGuy]

So basically, only pick Southern Blotting for paternity disputes (i.e. RFLPs), correct? Is S. blotting still the technique of choice in forensics (i.e. VNTRs)?

So really, the question was asking "when do you sequence DNA?" The answer being, "when you have a Dz involving a point mutation or 3-base pair substitution that does NOT affect a restriction site." They could get really picky if they expected you to know which diseases affected restriction sites 😱

I got hammered on this question because it expected you to know that CF is caused by hundreds of different 3-base pair substitutions...I ASSumed all cases were caused by Phe508 substitution.

Not exactly. The general idea is that Southern Blotting is not that accurate for small variations in DNA. It's useful for detecting different alleles in the same locus. PCR is good for known mutations. The general CF screening test detects 25 of the 1006 known mutations, but this identifies up to 97% carriers in certain populations.

 

[mjl1717]

Great post! 👍