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How to draw a blot from a RFLP physical map?

Genetics as it applies to evolution, molecular biology, and medical aspects.

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How to draw a blot from a RFLP physical map?

Postby ssimon10 » Wed Nov 23, 2011 2:43 pm

Here is the physical map of a region of DNA you are mapping by RFLP analysis:

===================
(probe)

_____|<-1.5kb->|<--2kb-->|<-1kb->|<----4Kb---->|
SITE: 1 2 3 4 5


The vertical lines with the numbers at the bottom( the numbers are beneath the vertical lines ) represent restriction sites for the enzyme EcoRI.; Sites 3 and 4 are polymorphic; the others are not. The distances between EcoRI sites are given. The extent of the probe you are using to detect the RFLP is indicated by a thick horizontal line (at top). You cut DNA from different individuals with EcoRI, separate fragments by electrophoresis, blot to a membrane, and hybridize the blot to a labeled probe. Draw a picture of what the blot will look like when the DNA comes from individuals homozygous for the following haplotypes:

the probe is on sites 2,3,4

the distance between site 1 and 2 is 1.5kb

site 2 and 3 is 2 kb sites 3 and 4 is 1kb and sites 4 and 5 is 4 kb ...

HAPLOTYPE SITE 3 SITE 4
A present present
B present absent
C absent present
D absent absent




This is what I know about how to approach this problem: All of the areas in between the restriction sites are from different individuals, right? I know that we're looking to know more about the variations in DNA sequence, and although usually knowing the object of the problem helps a lot, it doesn't seem to be helping here. I just... don't know what to do with the information I have, I guess. In class, I thought I knew what a haplotype meant, but I don't really know what it would mean to be "homozygous for the following haplotypes" followed by +/- data for letters whose origin I'm not even sure of. I guess I just need somebody to help me sort out all this information and point me in the right direction as far as how to start.

I am unable to figure out how i can represent a physical map on a DOT blot kindly help me out i really don't understand how it looks like on the paper please help me out with this i shall be great full to you. Thanks you guys.


Please help I am very lost! Best to you all
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Postby JackBean » Thu Nov 24, 2011 1:07 pm

Hi,
all you need to understant is, whether there is the restriction site or is not. That tells you the table of haplotypes.Thus haplotype A has both EcoRI sites 3 and 4, while in haplotype the site 4 was mutated and thus the enzyme is not able to cut the DNA there, thus the DNA stays in one piece and makes 1 + 4 kbp fragment (the fragments between sites 3&4 and between sites 4&5).
However, human is not haploid, but diploid, thus you have two copies of your chromosomes. It can happen, that they both contain the same restriction sites (say haplotype B on both chromosomes, thus being homozygous for the haplotype B), but they do not have to. Each chromosome can contain different haplotypes and then you are heterozygous and contain e.g. haplotype A and haplotype D.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re: How to draw a blot from a RFLP physical map?

Postby ssimon10 » Fri Nov 25, 2011 5:03 am

How would the presence or absence of site one have on the results? Would it just add an extra band on the blot?
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Postby JackBean » Fri Nov 25, 2011 7:13 am

no, if is the site absence, two bands will disappear and one other (of the combined size of the previous two bands) will appear on the gel.
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Re: How to draw a blot from a RFLP physical map?

Postby ssimon10 » Fri Nov 25, 2011 4:29 pm

What is site one is present? Since the probe is not one size one i thought in the presence you would not see any new band and in the absnece you would not see any change from the origional blot. Thanks for all your help I bet I will have a question later
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Postby JackBean » Fri Nov 25, 2011 8:01 pm

where exactly does the probe binds? It's not much clear from your picture.
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Postby ssimon10 » Fri Nov 25, 2011 8:18 pm

The probe covers the 2kb, 1kb, and half of 4kb fragments so everything between restriction sites 2 through 3, 3 through 4 and half of 4 through 5. ( Is this more clear?) so here is what I came up with let me know if I am on the right track if you do not mind. for lane A there would be a band 4 kb in length and 2 kb in length ( 1.5 and 1 bands are invisible), lane B would have a band at 5 kb ( 1 +4kb), and 2 kb ( 1.5 would be invisible), Lane C would have one at 4 kb, 3 kb ( 2+1), and (1.5 invisible), lane D would have one at 7kb (4+1+2), and 1.5 (invisible).
The next questions asks what effect would the presence or absence of site one have on the results?
For this I am not to sure of, but since the probe does not hybridized to site one then I would think it would not make much of a difference.
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Postby JackBean » Fri Nov 25, 2011 9:28 pm

the 1 kbp fragment is visible, since it is covered with the probe!

If the fragment between sites 1 and 2 is not covered with the probe at all, the presence/absence of site 1 does not have any effect.
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