Gene cloning.docx

A clone is a genetically exact copy. Cloning is a technique that allows us to make many identical copies of DNA such as a gene. Why is gene cloning useful? To further study the function of gene or protein it encodes. Determine effects of mutations on that gene sequence Develop diagnostics. Produce...

A clone is a genetically exact copy. Cloning is a technique that allows us to make many identical copies of DNA such as a gene. Why is gene cloning useful? To further study the function of gene or protein it encodes. Determine effects of mutations on that gene sequence Develop diagnostics. Produce a protein product of that gene is large amounts such as vaccines. All of the above require DNA that’s pure, homogenous and in sufficient quanitity. Gene cloning requires vector- DNA molecule which is used to carry the genetic material into another cell where it can be replicated or expressed. Such as plasmids. A vector containing foreign DNA is called recombinant DNA. Suitable vectors should be: Independent from host chromosome- Copied by host cell- usually achieved by inserting the recombinant plasmid into a host cell that copies the DNA. Maintaining the recombinant vector throughout the population of cells. Allow for selection of those cells that contains the vector. Would be beneficial to have a way to detect those cells that only contains the recombinant DNA. How to select the appropriate cells containg the vector or the recombinant vector? For those cells that contain the vector= Antibiotic resistance- can have an antibiotic resistance gene present therefore can differentiate. Nutritional selection such as amino acids. For those contains the recombinant vector= Colorimetric assay: Blue-white screening. When looking for cells that contain the recombinant vector, recombinant DNA would have been inserted into a restriction site in the MCS of the plasmid that lies withtin the Lac Z gene. If the Lac Z is disrupted, it won’t produce the beta galactosidase enzyme therefore it cannot convert X-Gal lactose analog into a blue compound and stays white. The blue-white screening works because the Lac Z gene is distributed by the recombinant DNA and the beta galactosidase enzyme into made therefore all colonies are white. Those that do not contain recombinant DNA will turn blue as the Lac Z gene is not disrupted. Why are plasmids used? They are ideal because: Contains origin of replication which allows them to be replicated by bacterial host. Most plasmids contain defined copy numbers- the number of plasmids that are present within a cell are highly regulated. Contains a antibiotic resistance gene- allows those cells that contains the plasmid to grow in the presence of that antibiotic within the growth medium. Contains selectable marker- which allows to select those cells that have only taken up recombinant plasmid and not just the plasmid itself. Process of gene cloning 1) The plasmid DNA is cut/linearised. 2) Piece of foreign DNA is ligated to cut ends of the plasmid DNA- ligation 3) Recombinant plasmid is inserted into bacteria- transformation. 4) Bacterial colonies are then screened for presence of recombinant plasmid. Cutting the plasmid Enzymes called restriction endonucleases/ restriction enzymes. These enzymes recognise very specific sequences in DNA called palindromic sequences. Restriction enzyme induces a double stranded cut in the DNA. Cut ends of DNA have single stranded overhangs which can stick to complementary sequences hence called ‘Sticky Ends’ Some enzymes produce ‘Blunt ends’ due to cutting symmetrically at recognition site. Ends of cuts DNA are uniform. The cloning plasmids contains a group of unique restriction sties clustered together at one location= multiple cloning sites (MCS). Ligation The cut ends of plasmid can be stuck back together using ligase enzyme. Or the cut ends of plasmid can be stuck together with another DNA molecule containing complementary ‘sticky ends. Sometimes the cut section of the DNA has overhangs which are complementary to the plasmid, so it forms complementary base pairing. Ligase catalyses the formation of phosphodiester bonds. Transformation and selection The recombinant DNA is introduced into the bacterial cells via heat shock. Heat shock induces changes in the fluidity of the membrane and causes pores to form in the membrane to allow DNA to enter. Very inefficient process as only 1 in 10K cells takes up plasmid. Antibiotic selection Recombinant plasmid will replicate in the bacterial cell and transferred to new cells via cell division. Most cloning plasmids contain a antibiotic resistance gene which allows bacteria to survive in presence of that antibiotic. By growing the bacteria with that antibiotic, bacteria without that antibiotic resistance genes will die and the ones who do contain it will survive and grow. Therefore, any bacteria that grows on the colony contains the plasmid- every cell within the colony will contain exactly same recombinant plasmid therefore a CLONE. Secondary selection Not all bacterial colonies will contain the right recombinant plasmid. Allows to distinguish between non-recombinant and recombinant clones. Blue-white screening Both sticky end and blunt ends can ligate together but blunt end ligation is less efficient.