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This Concept Map, created with IHMC CmapTools, has information related to: 12.2 Methods in Gene Cloning, 12.2 Methods in Gene Cloning Stage 2 Insertion of the target gene into a vector d) The lacz gene sequence contains a restriction site for a specific restriction enzyme.If the target gene is inserted within the restriction site,then the lacZ gen will not function, 12.2 Methods in Gene Cloning Stage 3 Introduction of vector into a host a) The plasmids carrying the target gene (recombinant plasmids) must be introduced into a host cell,for example,bacteria E.coli through transformation. The mixture of two types of plasmids is mixed with E.coli in a medium containing calcium chloride, 12.2 Methods in Gene Cloning Stage 4 Cloning of the target gene by the host cell e) The white colonies with recombinant plasmids are then cultured in a large scale, 12.2 Methods in Gene Cloning Stage 4 Cloning of the target gene by the host cell d) The bacteria which contain recombinant plasmids will form wwhite colonies because the lacZ gene is not function, 12.2 Methods in Gene Cloning Stage 1 Isolation of the target gene c) the DNA extract is then mixed with a specific restriction enzyme to cut it into fragments of various lengths known as restriction fragment, 12.2 Methods in Gene Cloning Stage 4 Cloning of the target gene by the host cell a) Following the introduction of the recombinant plasmids, the bacteria E.coli are cultured in a medium containing ampicillin and the sugar,X-gal, 12.2 Methods in Gene Cloning Stage 1 Isolation of the target gene a) can be isolated using two methods that is by cutting the gene from a complex chromosome using restriction enzymes or by producing complimentary DNA(cDNA), 12.2 Methods in Gene Cloning Stage 1 Isolation of the target gene d) the target gene that is insulin gene has to be seperated from the other restriction fragment.This can be done by using gel electrophoresis and a DNA probe.Gel electrophoresis seperates the various fragments of DNA according to their sizes.ADNA probe is then used to locate the target gene, 12.2 Methods in Gene Cloning Stage 3 Introduction of vector into a host c) This will result in three type of bacteria 1:bacteria that do not take up any plasmids 2:bacteria that take up non-recombinant plasmids 3:bacteria that take up recombinant plasmids, 12.2 Methods in Gene Cloning Stage 4 Cloning of the target gene by the host cell b) The transformed E.coli which has taken up plasmids will be able to grow and form colonies on the medium because they are resistant to ampicillin, 12.2 Methods in Gene Cloning Stage 2 Insertion of the target gene into a vector f) The plasmids which are now in the form of opened rings with sticky ends are mixed with fragments of the target gene, 12.2 Methods in Gene Cloning Stage 4 Cloning of the target gene by the host cell c) The bacteria that contain plasmids with intact lacZ genes (do not carry target gene) are able to produce the enzyme B-galactosidase which can hydrolyse X-gal.This will pro produce a compound which make the colony blue, 12.2 Methods in Gene Cloning Stage 3 Introduction of vector into a host b) Only a small amount of the bacteria will take up plasmids from the medium and from this,a lesser amount will contain the recombinant plasmids, 12.2 Methods in Gene Cloning Stage 2 Insertion of the target gene into a vector i) As the result,two types of plasmids are produce 1:plasmids that do not carry the target gne due to reannealing of their own sticky ends (non-recombinant plasmids) 2:recombinant plasmids which carry the target gene, 12.2 Methods in Gene Cloning Stage 1 Isolation of the target gene g) The mRNA of the target gene is extracted from cells where the gene actively synthesises the relative protein.In this case,the mRNA is extracted from pancrease cells which actively produce the hormon insulin, 12.2 Methods in Gene Cloning Stage 2 Insertion of the target gene into a vector e) The bacterial plasmids which have been isolated from bacterial cells must be mixed with the same restriction enzyme used to cut the DNA molecule during the isolation of the target gene.This is to produce the same sequence of sticky ends., 12.2 Methods in Gene Cloning Stage 2 Insertion of the target gene into a vector a) The gene is then inserted into vectors (gene carriers) such as plasmids or bacteriophages DNAs resulting in recombinant DNAs,combination of DNA from two source, 12.2 Methods in Gene Cloning Stage 1 Isolation of the target gene b) for example,the cloning of the insulin gene.In the first method, the human DNA from the donor must first be extracted from the human cell that has been cultered in the laboratory., 12.2 Methods in Gene Cloning Stage 2 Insertion of the target gene into a vector b) The cloning vector used,for example,bacterial plasmids posses marker genes such as ampR and lacZ, 12.2 Methods in Gene Cloning Stage 4 Cloning of the target gene by the host cell f) The bacteria will divide to produce new identical bacterial cells(clones).Each time the bacterial cells divide,the recombinant plasmids will also replicate producing multiple copies of the target gene(the insulin gene).The insulin gene is expressed to produce insulin and collected for commersial use.