Genetic Engineering Genetic engineering is also referred to

Genetic Engineering • Genetic engineering is also referred to as recombinant DNA technology – new combinations of genetic material are produced by artificially copying a piece from one organism and joining this copy with DNA of another organism (this works because the genetic code is universal) – the following must be accomplished to create new genetic combinations: 1. 2. 3. 4. locate a specific gene in the donor cell isolate this gene in a piece of donor DNA modify the donor DNA in a highly selective way transfer the modified donor DNA into host cells in a way that the gene will be expressed

Locating and isolating genes – can be accomplished using various techniques • Genetic probes – can be used if at least part of the DNA base sequence in the required gene is known – probe consists of a single strand of DNA that contains the known sequence of bases – probe is labeled with a radioactive marker – bases in genetic probe combine with the complementary bases on the donor DNA, revealing position of gene

• Reverse transcription – can be used when cells produce large amounts of the particular polypeptide 1. cells will contain m. RNA for polypeptide 2. m. RNA is isolated and can be used to make the complementary DNA through reverse transcription – reverse of normal transcription 3. requires DNA nucleotides and enzymes called reverse transcriptases 4. after m. RNA has been used to make DNA, m. RNA is removed and the complementary strand of DNA is made by adding the enzyme, DNA polymerase and more DNA nucleotides 5. the result is a double-stranded DNA molecule identical to the original DNA molecule 6. reverse transcriptases were first obtained from retroviruses

• Artificial DNA synthesis – a piece of DNA coding for a particular protein can be made artificially if the base sequence is known – this can be obtained from knowing the amino acid sequence of the protein – useful for small proteins

Enzymes – two important enzymes used in genetic engineering are restriction endonucleases and DNA ligases • Restriction endonucleases – cut DNA at specific points – cut ends are called “sticky ends” because they have exposed bases that will form hydrogen bonds with complimentary sticky ends from other DNA molecules cut by same restriction endonuclease • DNA ligases – these enzymes stick lengths of DNA together • genetic engineers are able to use both enzymes to “cut and splice” DNA

A DNA vector is used to transfer DNA from one cell to another • most commonly used vector is a plasmid – small ring of DNA contain in some bacteria • separate from main chromosome • replicate independently • can be passed from one bacterial cell to another (occurs naturally) • transfer to another cell can be encouraged artificially • restriction enzymes and DNA ligase are used to cut open a plasmid so that donor DNA (gene) can be inserted • modified plasmid is transferred to another living cell (usually a bacterial cell) • this process is possible because of the universality of the genetic code

Genomic Libraries – (total DNA of a cell is referred to as a genome) • genomic library – a collection of DNA fragments that are more or less representative of all the DNA in the genome • each fragment is spliced into a plasmid, which is usually contained inside a bacterial cell • restriction enzymes may divide a single gene into more than one fragment, therefore some genes in a genome library may be divided up among two or more clones (bacterial cell containing plasmid)