Hi,
This might be a naive question, but it has been troubling me for a bit. I have the human genome (hg19) cut by 2 restriction enzymes and I have a list of cut sites. I am assuming that this is by default the +ve strand of the DNA. Now I have a gene whose location is chr11 355953 357144 - meaning, it is on the -ve strand. I want to find those cut sites which are just before and after this gene (and also those that are within this gene).
My confusion is that can I simply transpose these coordinates on the -ve strand as it is to the positive, making it chr11 355953 356144 + and then find sites before and after this from the list of cut sites? Or will the transformed coordinates on the +ve strand be different (for example, taking the length of the chromosome into account)?
I am just not sure that if I simply use the -ve strand coordinates on the +ve strand, then would the same cut sites be valid. The restriction enzymes are palindromic.
Thank you!
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written
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c_u
1 answer
Hi, welcome.
To make your restriction map:
a) consider the coordinates of your gene irrespective of its sense strand, i.e. chr11:355953-356144
b) it is useful to expand its coordinates a bit both upstream and downstream (because you are interested in restriction sites surrounding it); say, we expand its coordinates by 10 Kb; you would get chr11:345953-366144
c) download the + strand of this genomic region, i.e. chr11:345953-366144:+
d) the 3'-end (the "feet") of the sense strand of your gene will point to the left, because your gene (actually, its sense strand) is on the genomic - strand
e) find occurrences of all restriction sites of your interest, for both enzymes; note that each site would have a start position and an end position. Say, you have a site with coordinates chr11:355953-355958 (corresponding in your map to positions 10001-10006, a 6bp-long site). You can now read its sequence on the plus strand (say, "AAAAAT") or on the minus strand (it would read "TTTTTA"). Of course, our local coordinates (10001-10006) mark positions counting from the left-end of the map, simply because this is the standard procedure! When we want to refer to "TTTTTA" we would simply write 10001-10006 - strand (or, genomically speaking, chr11:355953-355958:-), rather than defining a new coordinate system that starts counting from the right-end of the map.
Also note that normally resitriction sites are palindromic sequences (so that a real example might be "ACTAGT" for both strands).
Hope this helps!
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It usually helps your reasoning to draw, a chromosome, a gene in both directions, and the identified sites.
Thanks @Wouter for the reply. What I am confused is if the restriction sites on the positive strand would map to the same locations on the -ve strand too. Believe me, I have drawn quite a few diagrams since yesterday :)
I am also often confused. The best way for me is to use UCSC genome browser and its Restr_enzyme track. You can try UCSC genome browser-> human -> hg19 and then click hide all. Then add custom track and paste
chr11 355953 356144 TestGene 1000 +Then in mapping and sequencing click on restr-enzyme track and give a comma separated list of enzymes. These will be displayed along with the coordinates of your bed.