Blog. 18 December Prezi Awards The best presentations have arrived. 5 December Do this, not that: Keynote speech. 28 November Wady i Zalety Klonowania Idea 1. Idea 2. Rozmnażanie bezpłciowe – naturalne klonowanie. Klonowanie zachodzi przez: Klonowanie DNA. KLONOWANIE Klony DNA służą do: Co to jest klonowanie? Klonowanie – tworzenie genetycznej kopii fragmentu DNA, komórki lub organizmu.
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Enzymy restrykcyjne i ligaza DNA. For instance, the human insulin gene is expressed in E.
File:Gene cloning PL.svg
And plasmids tend to be circular DNA so we will paste it into a plasmid. Actually, let me just draw, let me just try to draw the two strands just so we remind ourselves.
Plasmid cut with the same restriction enzyme at a site following a promoter for bacterial expression. So you would see things like this, olonowanie would be many, many, many cells of bacteria, there would be colonies of bacteria. Repeated nebulisation of non-viral CFTR gene therapy in patients with cystic fibrosis: So that is DNA ligase, which klonowqnie can think of it as helping to do, helping to do the pasting. After transformation, bacteria are selected on antibiotic plates.
Each colony starts from a single bacterium with a single plasmid, so all the bacteria in a colony with have the same plasmid either “good” or “bad”. Each surviving bacterium will give rise to a dn, dot-like group, or colonyof identical bacteria that all carry the same plasmid.
Klonowanie i rekombinacja DNA
And we also saw DNA ligase when we studied replication. And as it reproduces it also is reproducing the plasmids and because it has this antibiotic resistance it is going to grow on this nutrient antibiotic mixture and the other bacteria that did not take up the plasmids are not going to grow. So there we go. In a typical DNA cloning procedure, the gene or other DNA fragment of interest perhaps a gene for a medically important human protein is first inserted into a circular piece of DNA called a plasmid.
Enzymy restrykcyjne i ligaza DNA. Now the next question, and I’m over simplifying things fairly dramatically is well you now have a bunch of bacteria that have a bunch of copies of that gene, how do you make use of it? Let’s take a closer look at each step.
It appears that the heat shock causes the formation of pores in the bacterial membrane, through which the DNA molecules can pass. For instance, when we try to insert a gene into a plasmid using a particular restriction enzyme, we may get some cases where the plasmid closes back up without taking in the geneand other cases where the gene goes in backwards.
A plasmid typically contains an antibiotic resistance genewhich allows bacteria to survive in the presence of a specific antibiotic. Why does it matter if a gene goes into a plasmid backwards? Protein production and purification. The copies are often made in bacteria.
What we want to get is:. It’s going to take in the plasmid. The purified protein can be used for experiments or, in the case of insulin, administered to patients.
Because of these possibilities, it’s important to collect plasmid DNA from each colony and check to see if it matches the plasmid we were trying to build. The antibody in the column is designed to bind to our protein of interest, and not to any other molecules in the mixture. How can pieces of DNA from different sources be nda together? Klonoowanie two molecules have matching overhangs, they can base-pair and stick together.
File:Gene cloning – Wikimedia Commons
Well, the first thing we wanna do is we wanna cut this gene out some how. So you don’t want that one. Klonowanie i rekombinacja DNA. Grow up lots of plasmid-carrying bacteria and use them as “factories” to make the protein. There are many other proteins and macromolecules floating around in bacteria besides the target protein e. In a typical cloning experiment, a target gene is inserted into a circular piece of DNA called a plasmid.
The bacteria in the large culture are induced to express the target gene through addition of a chemical signal to the culture medium.
Thus, bacteria that took up the plasmid can be selected on nutrient plates containing the antibiotic.
Expression of the gene leads to production of mRNA, which is translated into protein. This step uses restriction enzymes and DNA ligase and is called a ligation.
Klonowanie i rekombinacja DNA (film) | Khan Academy
And in fact that’s what would give the bacteria its antibiotic resistance but if this gene was say for insulin, well then the bacteria will produce klonowaie bunch of insulin, a bunch of insulin molecules, which you might be able to use in some way. Cutting and pasting DNA.
So let me label these. Other examples of recombinant proteins include human growth hormone, which is given to patients who are unable to dba the hormone, and tissue plasminogen activator tPAwhich is used to treat strokes and prevent blood clots. This is not a useful plasmid if we want to express the gene in bacteria.
Not all colonies will necessarily contain the right plasmid. Many restriction enzymes produce cut ends with short, single-stranded overhangs.