gene cloning-the use of vectors Flashcards
what is in vivo gene cloning?
-in vivo, means transferring the DNA fragments to a host cell using a vector
What is in vitro cloning ?
- in vitro, using the polymerase chain reaction
- DNA is inserted by introducing the DNA into bacteria cells by transformation
How do you use the insertion of DNA fragments into a vector?
-the preparation of the DNA fragment involved the addition of extra lengths of DNA
-for the transcription of any gene to take place, the enzyme that synthesises mRNA (RNA polymerase) must attach to the DNA near a gene
-the binding site for RNA polymerase is a region of DNA, known as promoter
-the nucleotide bases of the promoter attach both RNA polymerase and transcription factors and so begin the process of transcription
-if we want out DNA fragment to transcribe mRNA in order to make a protein, it is essential that we attach ti it the necessary promoter region ot start the process
-in the same region of DNA binds RNA polymerase and begins transcription of a gene, another region releases RNA polymerase and ends transcription of a gene, another region releases RNA polymerase and ends transcription
-this region of DNA is called a terminator
-again we need to add a terminator region to the other end of our DNA fragment to stop transcription at the appropriate pointy
-only a few bacterial cells (as few as 1%) take up the plasmids when the two are mixed together
-some plasmids will have closed up again without incorporating the DNA fragment
-sometimes the DNA fragment ends join together to form its own plasmid once an appropriate fragment of DNA has been cut from the rest of the DNA and the promoter and terminator regions added, the next task is to join it into carrying unit, known as a vector
-this vector is used to transport the DNA into the host cell
-there are different types of vector but the most commonly used in the plasmid
-plasmids are of vector but the most commonly used is the plasmid
-plasmids are circular lengths of DNA, found in bacteria, which are separate from the main bacterial DNA
-plasmids almost always contain genes for antibiotic resistance and restriction endonucleases are used at one of these antibiotic-resistance genes to break the plasmid loop
-the restriction endonuclease used is the same as the one that cut out the DNA fragment
-this ensured that the sticky ends of the opened-up plasmid are complementary to the sticky ends of the DNA fragment
-when the DNA fragments are mixed with the opened-up plasmids, they may become incorporated into them
-where they are incorporated, the join is made permanent using they enzyme DNA ligase
-these plasmids now have recombinant DNA
-the sequence of DNA that are cut by restriction endonucleases are called recognition sites
-if the recognition site is city in a staggered fashion, the cut ends of the DNA double strand are left with a single strand which is a few nucleotide bases long
-the nucleotides on the single strand at one side of the cut are obviously complementary to those at the other side because they were previously paired together
-if the same restriction endonuclease is used to cut DNA, then all the fragments produced will have ends that are complementary to one another
-this means that the single-stranded end of any one fragment can be joined (stuck) to he single-stranded end of any other fragment
-in other words, their ends are sticky
-once the complementary bases of two sticky ends have paired up, an enzyme called DNA ligase is used to bind the phosphate-sugar framework or the two sections of DNA and so unity them as one
-sticky ends have considerable importance because, provided the same restriction endonuclease is used, we can combine the DNA of one organism with that of any other organism
once the DNA has been incorporated into at least some of the plasmids, they must then be reintroduced into bacterial cells
-this process is called transformation and involved the plasmids and bacterial cells being mixed together in a medium containing calcium ions
-the calcium ions and changes in temperature, make the bacterial membrane permeable, allowing the plasmids to pass through the cell-surface membrane into the cytoplasm
-however, not all the bacterial cells will possess the DNA fragments with the desired gene for the desired protein
-the first task is to identify which bacterial cells have taken up the plasmid
-one way to do this is to use the fact that bacteria have evolved mechanisms for resisting the effects of antibiotics, typically by producing an enzyme that breaks down the antibiotic before it can destroy the bacterium
-the genes for the production of these enzymes are found in the plasmids
-some plasmids carry genes for resistance to more than one antibiotic
-one example is the R-plasmid, which carries genes for resistance to two antibiotics, ampicillin and tetracycline
-the task of finding out which bacterial cells have taken up the plasmids entails using the gene for antibiotic resistance, which is unaffected by the introduction of the new gene
-like the gene for resistance to ampicillin
-this is an effective method for showing which of the bacterial cells have taken up in plasmids
-however some cells will have taken up the plasmids
-however, some cells will have taken up the plasmids and then closed up without incorporating the new gene, and these will also have survived
-the next task is to identify the cells without the new gene and eliminate them
-this is achieved using marker genes
What ampicillan resistance marker genes?
- all the bacterial calls are grown on a medium that contains the antibiotic ampicillin
- bacterial cells that have taken up the plasmids will have acquired the gene for ampicillin resistance
- these bacterial cells are able to break down the ampicillin and therefore survive
- the bacterial cells that have not taken up the plasmids will not be resistant to ampicillin and therefore die
What are antibiotic-resistance marker genes?
- the use of antibiotic-resistance genes as markers is rather old technology and has been superseded by other methods
- however, it is an interesting example of how science works, particularly of the way in which scientists use knowledge and understanding to solve new problems, use appropriate methodology and carry out relevant experiments
- to identify those cells with plasmids that have taken p the new gene we use a technique called replica plating
- this process uses the other antibiotic-resistance gene in the plasmid: the gene that was. Cut in order to incorporate the required gene
- like the gene for resistance to tetracycline
- as this gene has been cut, it wild no longer produce the enzyme that breaks down tetracycline
- in other words, the bacteria that have taken up the required gene will no longer be resistant to tetracycline
- we can therefore identify these bacteria by growing them on a culture that contains tetracycline
- the problem is that treatment with tetracycline will destroy the very cells that contain the required gene
- however by using a technique called replica plating it is possible to identify living colonies of bacteria containing the required gene
How are fluorescent markers added to DNA?
- a more recent and more rapid method is the transfer of a gene from a jellyfish into the plasmid
- the gene in question produced a green florescent protein (GFP)
- the gene to be cloned is transplanted into the centre of the GFP gene
- any bacterial cell that has taken up the plasmid with the gene that is to be cloned will not be able to produce GFP and to flouresce
- unlike the cells that have not taken up the gene, these cells that have taken it up will not flouresce
- as the bacterial cells with the desired gene are not killed, there is no need for replica plating
- result can be obtained by simply viewing the cells under the microscope and retaining those that do not flouresce which makes the process more rapid
What are lactas enzyme markers?
- another gene marker is the gene that produces the enzyme lactase
- lactase will turn a particular colourless substrate blue
- again, the required gene is transplanted into the gene that makes lactase
- if a plasmid with the required gene is present in a bacterial cell, the colonies grown from it will not produce lactose
- therefore, when these bacterial cells are grown on the colourless substrate they will be unable to change its colour
- when the gene has not been taken up by the bacteria, they will not turn the substrate blue
- these bacteria can be discounted
