Title: Genetic Techniques 1 Learning question: What are the applications of genetic engineering? Starter: Can you think of any real life applications of genetic engineering?

Learning Objectives (a) outline the function of restriction enzymes (restriction endonucleases) and ligase enzymes in separating and joining specific DNA sequences; (b) describe, with the aid of diagrams, the palindromic nature of restriction enzyme recognition sequences;

Key words Genetic engineering Vector Cloning Restriction enzymes Blunt ends Sticky ends Annealing Restriction fragments DNA ligase Palindromic sequence

The role of restriction and ligase enzymes Genetic engineering – replace, modify or add DNA coding sequences to a living organism. Uses enzymes to “cut” up parts of DNA from one organism and “stick” them into another. Resulting new organism will incorporate genes and therefore proteins from imported DNA.

The role of restriction and ligase enzymes 3 main stages to GE: Isolating and identifying gene> protein Putting isolated gene into another organism using a vector Cloning the organism to make several copies of the imported gene product.

The role of restriction and ligase enzymes Restriction enzymes (restriction endonucleases) cut DNA at particular sequences of bases. Two types: Enzymes that cut straight across – blunt ends Enzymes that cut in a staggered way – sticky ends (more useful)

Sticky ends Short stretches of single stranded DNA are complementary to each other. If both ends are cut with the same enzyme, the sticky ends will stick together by complementary base paring, forming hydrogen bonds Annealing is the name given to the process by which sticky ends form hydrogen bonds.

Restriction fragments DNA has several recognition sites where restriction enzymes can “cut” at very specific points. These restriction sites are only 4-8 base pairs long, so they are specific sites. Cuts at restriction sites produces lots of DNA fragments of different lengths. The resulting fragments of DNA are known as restriction fragments.

Bacteria and DNA Restriction enzymes are produced naturally by bacteria as a defence mechanism against viruses. The restrict viral growth Restriction enzymes used in GE are named after the bacteria it came from, e.g. EcoR1 – e.coli strain R was the first to be identified.

Cutting and pasting Once DNA fragments have been annealed to DNA of another organism, the broken DNA needs to be repaired. Remember annealing is the formation of complementary base pairs – this is done by weak hydrogen bonds. DNA ligase enzymes repair broken DNA by forming covalent bonds between the new sections.

Palindromic recognition sites Words or phrases that can be read the same in both directions are described as palindromic Level, Hannah etc Restriction enzymes cut DNA at palindromic sequences e.g. GGCCCCGG

Describe the stages of genetic engineering in this diagram restriction endonuclease enzymes ligase enzymes Describe the stages of genetic engineering in this diagram

Recombinant DNA Technology The desired gene is _________ in human Bacterial cell with a __________ selected isolated plasmid Desired Gene cut out of Chromosome using __________ _________ Plasmid removed and cut Open using ________ __________. restriction restriction endonuclease endonuclease Gene sealed in Plasmid using ____. Which joins ______ ends together. ligase plasmid

Recombinant DNA Technology Plasmid inserted into ________ _______ _______ host bacterium ______ _______ Used to check the gene is present DNA probe cell Recombinant bacteria Allowed to ________ rapidly Bacteria ________ that _______ the gene as a protein which is then _________. identified multiply expresses propagated Human proteins produced by this method include _____ (trade name humulin) , _____ ______ ______ (HGH)

Marker genes Only a small percentage of cells take up the recombinant DNA. Identifying these cells is important. This can be done with marker genes. Marker genes, when expressed, display easily observable characteristics. When recombinant DNA is transferred into a vector of the host cell, the marker gene goes with it. This means that if we can see the expression of MG we know that GE has been succesful.

Antibiotic resistance markers and reporter systems Marker genes can code for resistance to particular antibiotics. GE organisms grown in a growth medium containing antibiotics will do this. Only GE organisms will grown in this environment as the antibiotic will kill other organisms that fail to take up the plasmid. This technique has worked well, but there is concern about spreading “resistant genes”.

Antibiotic resistance markers and reporter systems Reporter systems have superseded antibiotic resistant genes. This involves bioluminescence – target genes will fluoresce, allowing them to be followed/isolated

Another route to isolating a gene 20000-30000 protein coding genes in the human genome. Isolating specific genes is difficult – complementary DNA technique is used Complementary DNA (cDNA) is copied from mRNA The enzyme reverse transcriptase synthesises DNA from an RNA template. Certain retroviruses have this enzyme capable of doing this. DNA produced is single stranded, but DNA polymerase can be used to produce a double stranded cDNA “gene”. Beta cells in the pancreas make the hormone insulin, therefore manufactures mRNA that codes for insulin. mRNA can be isolated and used to make cDNA for the human insulin gene.

Another route to isolating a gene Advantages of GE using bacteria Microorganisms are simple and easy to use Quick and easy to culture Few ethical issues about their use Disadvantages of GE using bacteria Cannot make human proteins Animals can be used, but products have to be extracted via milk/urine and not directly from cells Plants can be used – products secreted via roots.