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2. Gene Technology or Recombinant DNA Technology is about the manipulation of genes Recombinant DNA Technology involves the isolation of DNA sequences from one organism and combining them with the DNA from a different organism This technique allows organisms to express genes and thus manufacture proteins that they would not normally produce – their DNA has been engineered to manufacture a product that is foreign to their natural genetic make-up The procedure of Recombinant DNA Technology involves introducing segments of DNA (genes) into a HOST by means of a carrier (VECTOR) system The foreign DNA (gene) becomes a permanent feature of the host such that, during replication of the host cell, the gene is also passed on to its daughter cells along with the rest of its own DNA – the foreign gene is therefore cloned The manufacture of human insulin and growth hormone are examples of the application of Recombinant DNA Technology Gene Technology

3. Obtain the gene that codes for the desired protein product Select a suitable vector (carrier) for transferring the gene to the host cells; the most commonly used vectors are bacterial plasmids and viruses Insert the selected gene into the chosen vector Allow the vector to carry the gene into the host cells; commonly used host cells are bacteria and yeast cells – they are chosen for their rapid rates of reproduction and allow the gene to be CLONED Locate the host cells that have successfully taken up the desired gene Culture the transformed cells on a commercial scale using large fermenters Isolate and purify the desired ‘gene product’ – Downstream Processing The Procedure of Recombinant DNA Technology

4. The genetic engineer uses five basic ‘tools’ during the procedure of Recombinant DNA Technology A host organism that will take up the vector containing the gene and reproduce rapidly in order to supply many copies of the gene (gene cloning) – the bacterium E. coli is commonly used as a host Vectors (carriers) into which the desired gene may be inserted and which are capable of carrying the gene into a suitable host – bacterial plasmids are commonly used as vectors circular plasmid An enzyme capable of ‘glueing’ an isolated gene into a ‘cut’ vector – DNA ligase is responsible for re-forming the DNA backbone following insertion of the gene plasmid with inserted gene Isolated genes that code for the desired product Enzymes capable of ‘cutting’ DNA at specific sites – these enzymes are known as Restriction Enzymes The ‘Toolkit’

5. Restriction enzymes, also known as Restriction Endonucleases, are a group of enzymes found in bacteria that recognise specific DNA sequences of four to six nucleotides and make their incision within that sequence The specific nucleotide sequences, recognised by restriction enzymes, are called restriction sites and these are usually in the form of palindromes Palindromes are nucleotide sequences that are symmetrical, about an axis, and read the same in opposite directions in the two strands of DNA portion of double- stranded DNA central axis A restriction enzyme known as Eco R1, makes double-stranded cuts between the A and G nucleotides on either side of the central axis enzyme cuts The cuts from this enzyme are staggered and produce single-stranded regions called ‘sticky ends’ ‘sticky ends’ Restriction Enzymes are the engineer’s DNA – cutting scissors The ‘Toolkit’

6. Some restriction enzymes, such as Hpal, cut the DNA strands at positions directly opposite one another, giving blunt ends to the fragments Hpal recognises the nucleotide sequence GTTAAC and ‘cuts’ between the T and A nucleotides about the central axis enzyme cuts blunt ends Over seven hundred different restriction enzymes have now been identified and isolated from bacterial cells; each enzyme is named after the bacterial strain from which it was derived Eco R1 is from Escherichia coli, strain RY13 Bam H1 is from Bacillus amyloliquefaciens, strain H The ‘Toolkit’

7. Restriction enzymes that generate ‘sticky ends’ are very useful tools to the genetic engineer The same restriction enzyme recognises the same nucleotide sequence in the DNA from different species and creates the same ‘sticky ends’ When the DNA fragments from the two different species are mixed together, the complementary bases of their ‘sticky ends’ will be attracted to one another and form hydrogen bonds In this way, DNA fragments from different sources can be brought together and joined DNA ligase is the enzyme that seals fragments of DNA together The ‘Toolkit’

8. Fragment of DNA from species X Fragment of DNA from species Y Complementary sticky ends created by cutting the DNA from each species with the same restriction enzyme Complementary bases on the sticky ends of the DNA from the different species are attracted to one another Hydrogen bonds form between the bases and the enzyme DNA ligase seals the sugar- phosphate backbone of the DNA molecule Recombinant DNA is formed

9. VECTORS Vectors are carrier DNA molecules into which DNA fragments containing specific genes can be inserted Vectors are the means by which selected genes are carried into host cells where the desired gene is then cloned The isolated plasmids of bacterial cells and the DNA of bacteriophages (viruses that infect bacteria) are frequently used as vectors Plasmids are small, circular, self-replicating double-stranded DNA molecules found in bacterial cells,which are separate from the main bacterial chromosome main chromosome plasmid The ‘Toolkit’

10. Genes coding for ‘desirable products’ can be spliced into plasmids to form RECOMBINANT PLASMIDS When these plasmids are taken up by bacterial host cells, they replicate along with the host cell and clone the desired gene Plasmids are obtained from cultures of bacterial cells; bacterial cells are broken open and the plasmids are separated out by centrifugation Homogenised bacterial cells, when subjected to centrifugation, provide the plasmids into which foreign genes can be inserted The ‘Toolkit’

11. Total human DNA extracted from human cells - known as genomic DNA Plasmid The two DNA molecules are attracted to one another and, in the presence of DNA ligase, form a recombinant DNA molecule Both the plasmid and the human DNA are treated with the SAME restriction enzyme so that the DNA from both sources will have complementary ‘sticky ends’ DNA fragment with sticky ends complementary to those on the ‘cut’ plasmid Recombinant plasmid Making Recombinant Plasmids

12. When host bacterial cells are mixed with these recombinant plasmids, they may take them up and become transformed; these bacterial cells are now described as transgenic organisms as they contain and express the genetic material from a different species When this transformed bacterial cell divides, the recombinant plasmid replicates and copies of the plasmid (containing the foreign DNA) are passed to the daughter cells The foreign DNA has been cloned