Question: Sequencing plasmid with duplicated region
0
gravatar for Fman
4.5 years ago by
Fman0
Belgium
Fman0 wrote:

Dear all,

I am trying to sequence a plasmid, but I get some weird results and it seems that a region has been duplicated.

Now I am wondering: what approach might be the best in order to try and sequence in completely? Or is it pointless?

Now I have 2 contigs and by using primer walking I tried to extent the contigs in order to link them, but what happens is that rather than extending it, the new sequences are just "placed" inside the other contig or they are added to the other side of the contig. It does not extent the contig!

It is pretty weird.

 

any insights?

Its sanger sequencing, 1 plasmid.

sequencing • 1.6k views
ADD COMMENTlink modified 4.5 years ago by Whetting1.5k • written 4.5 years ago by Fman0
2
gravatar for Whetting
4.5 years ago by
Whetting1.5k
Bethesda, MD
Whetting1.5k wrote:

how big is the duplication? too long for a single run?

I would start with good old restriction digest mapping. find a restriction fragment(s) that contains the duplication. Design primers ans only sequence this fragment...

ADD COMMENTlink written 4.5 years ago by Whetting1.5k

Not sure I understand you. You would remove the part with the duplication and just sequence this?

 

The duplication is (as far as I can trust the results so far) 373 basepairs, but I think its bigger, I have to check it more.. There is something really weird going on with the plasmid.

ADD REPLYlink written 4.5 years ago by Fman0

what evidence do you have of a duplication? Just the sanger reads? When you do a restriction digest, and compare it to the parental plasmid, you should be able to figure out how much bigger it is. Through the use of a couple different enzymes, you will be able to zoom in on the region where the extra DNA is inserted. Once you know this it should be really easy to design primers and sequence the insert....
 

ADD REPLYlink written 4.5 years ago by Whetting1.5k

I see your point. But this will mean a lot of work! I also do not know we have the needed restriction enzymes to cut it up.

BTW: this is the parental plasmid! Its just a plasmid that I want to use, so its the parental plasmid itself ... I have not inserted anything...

And yes: the only evidence I have are the sanger reads that I tried to align to eachother using programs... I am not an expert on this type of work so perhaps I am making mistakes (I am just using the programs to align the reads, thats it and now I am trying to figure out whats going wrong).

 

ADD REPLYlink written 4.5 years ago by Fman0

it is not that much work. Digestion + gel will take 10' hands on work. You went through the effort of cloning and sequencing...

ADD REPLYlink written 4.5 years ago by Whetting1.5k

Yeah, thats true, I might concider it... But in short: I need to digest it and then have the digested part sequenced? Or ?

ADD REPLYlink written 4.5 years ago by Fman0

1) digest with a couple of enzymes

2) figure out, based on the digest pattern, where the extra DNA is located

3) sequence that fragment

However, if the fragment is really only 400bp, there should be plenty of DNA flanking the duplication. This should allow you to figure it out?

ADD REPLYlink written 4.5 years ago by Whetting1.5k

But to be honest: the fragment with the duplication in it.. I am pretty sure I already sequenced it (not just the duplication, but much more, further parts).. with long runs! I already sequenced with many many primers (30 or something) so one should think it should already be enough to link the parts...

Its just a very very weird result!

 

ADD REPLYlink modified 4.5 years ago • written 4.5 years ago by Fman0

I think you are counting on these programs too much (I know this is a bioninformatics site, but you need to understand what they are doing). Where did you get this plasmid? Do you have the sequence prior to the insertion?

ADD REPLYlink modified 4.5 years ago • written 4.5 years ago by Whetting1.5k

I am not a bio-informaticus expert so yeah, I do use those programs, but I also looked at the contigs and checked whether they make sense and tried to make a more correct config out if it, but it does not make sense.

Its just a plasmid we have. There is no sequence before the insertion. All I have is an old sequence (that seems to be incorrect anyway) and some general information.

After looking into the sequences and leaving some out to make a more correct alignment.. still nothing! The sequence makes no sense.

 

ADD REPLYlink written 4.5 years ago by Fman0

I can have a look at your sequences if you want to...shoot me an email and I can see whether I can help...

ADD REPLYlink written 4.5 years ago by Whetting1.5k

That would be nice! But how do I send you an email? I can not seem to figure it out... Perhaps its because I am a new member that I can not see your email? 

ADD REPLYlink written 4.5 years ago by Fman0

Hallon

how can I send you the sequence files?

ADD REPLYlink written 4.4 years ago by Fman0
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