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How is a specific gene removed from DNA

by Biosyn
Tags: dna, gene, inaz, pseudomonas syringae
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Biosyn
#1
Feb20-12, 12:11 PM
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I was reading an article on Pseudomonas syringae and how removing a gene called inaZ from it's DNA will allow it to be sprayed on crops to prevent the crops from freezing during the winter.


How is the inaZ gene removed from the DNA? I'm thinking that they use restriction enzymes that cut out the specific sequence of the gene, and then ...I'm not sure how the DNA is put back together. Does a DNA ligase just come around and bind the two ends together?

What restriction enzyme is used on Pseudomonas syringae dna?

Please correct me, thanks!
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MrRagnarok
#2
Feb22-12, 04:30 PM
P: 16
Once you know the sequence surrounding your favorite gene you can indeed find restriction sites surrounding it and excise the gene. You can then take this excised gene and ligate it to a receptive vector (like a bacterial plasmid).

Depending on which restriction enzymes you used originally (there are thousands of them) your gene will have overhanging nucleotides at its ends. Using these 'sticky' ends you match the gene to a vector with complementary sticky ends. A ligase is used to cement the phosphate backbone of the gene to the plasmid.


Your Gene
TACANNNNCGA
....GTNNNNGCTCG

(Ignore the periods, the formatting will not work if I use spaces. N=any nucleotide)

Notice the overhang TA- and -CG.
The TA will naturally bind to a prepared vector with a AT overhang
The CG will naturally bind to a prepared vector with a GC overhang

So a vector (like a bacterial plasmid) is made to have complementary overhangs and the gene automatically base pairs with it.

Circular -NNN.....TACANNNNCGA.....GCNNN- Circular
Plasmid -NNNAT.....GTNNNNGCTCG.....NNN- Plasmid

(Again ignore the periods, treat them as blanks)

DNA ligase is used to connect phosphate backbone (not pictured on this simple example) and the ligase completes the circular DNA so that there are no nicks in the DNA (which would weaken it).

Now this plasmid carrying your gene can be recognized by the bacteria and as the cell divides the bacteria will duplicate the gene (and the plasmid) with each division. In a very short period of time you will have billions of these genes.

I'm unfamiliar with plant genetics, but like I mentioned above there a literally thousands of restriction sites (in animal genetics) so the enzyme used depends on the size of the gene fragment you'd like.
Biosyn
#3
Feb22-12, 05:29 PM
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Thank you! :)

p.s. If I cut out part of the gene, will the rest automatically bind if there are sticky ends and the base pair matches?

Ygggdrasil
#4
Feb22-12, 06:24 PM
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How is a specific gene removed from DNA

There are a number of ways to modify genes in organisms, and the precise method to use depends on the organism being modified and the nature of the modification to the gene. Often, "removing" the function of a gene does not require physically removing the gene from the DNA of the organism. Rather, a simple mutation can be introduced into the coding sequence of the gene so that the gene product is not functional, or a mutation can be introduced into the regulatory sequences preceding the gene to ensure that the gene cannot be properly made. Such mutants can be created either by randomly mutagenizing the organism of interest and screening for organisms with a mutation in the gene of interest, or though more precise genetic engineering techniques. One common way of performing genetic engineering is through a technique called recombineering.
MrRagnarok
#5
Feb22-12, 07:30 PM
P: 16
Quote Quote by Biosyn View Post
p.s. If I cut out part of the gene, will the rest automatically bind if there are sticky ends and the base pair matches?
Yes, the base pairs bind readily due to hydrogen bonding but to form the phosphodiester bond, that is the flexible supportive backbone of the DNA molecule, you would need DNA ligase (and ATP).

ps Apparently, this thread is restricted to only Norse mythology themed profile names. So speak up Odin, Fenrir and Loki. I know you're out there
Biosyn
#6
Feb22-12, 09:46 PM
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Quote Quote by MrRagnarok View Post
Yes, the base pairs bind readily due to hydrogen bonding but to form the phosphodiester bond, that is the flexible supportive backbone of the DNA molecule, you would need DNA ligase (and ATP).

Got it! Thanks for your help!

ps Apparently, this thread is restricted to only Norse mythology themed profile names. So speak up Odin, Fenrir and Loki. I know you're out there
what?
my ignorance has compromised my ability to understand what you mean :P
Borek
#7
Feb23-12, 02:56 AM
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Quote Quote by Biosyn View Post
my ignorance has compromised my ability to understand what you mean :P
http://en.wikipedia.org/wiki/Yggdrasil

http://en.wikipedia.org/wiki/Ragnarok
MrRagnarok
#8
Feb23-12, 08:36 AM
P: 16
Quote Quote by Biosyn View Post
my ignorance has compromised my ability to understand what you mean :P
People are only ignorant when they stop asking questions.


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