Genetic Engineering Tools and Editing

What does a restriction endonuclease do

• enzymes (eg EcoR1) which cut DNA at a specific sequence of bases called a restriction site

• Make staggered cuts leaving unpaired single stranded ends

• Exposed bases form H-bonds to base pair with complementary sticky ends produced by same restriction enzyme

What is an example of a sequence recognised by restriction endonucleases

often palindromic 

What are the 3 ways by which genes can be obtained

• extracted directly from donor organism using restriction endonucleases

• Synthesised chemically from nucleotides

• Synthesised from the mRNA of a donor organism

  • Eg extract mRNA for insulin from the β cells of the islets of Langerhans in the pancreas

How can genes be obtained using mRNA

• extract correct mRNA identified by size (eg for insulin from the β cells of the islets of Langerhams in the pancreas)

• Use enzyme reverse transcriptase to produce a single stranded cDNA copy

• Then add DNA polymerase to make this double stranded.

How is a recombinant DNA formed 

• Recombinant DNA by joining pieces of DNA from two or more different sources

• Genes can be transferred from one species to another different species to form a transgenic organism because the genetic code is universal, so any gene can be expressed. 

• Transgenic organisms can produce a new protein on top of their existing proteome, gaining a new characteristic.

• DNA ligase catalyses the formation of phosphodiester bonds to seal the “gap” in sugar phosphate backbone creating the recombinant DNA.  

What is a plasmid and explain its structure

• small, circular piece of DNA which replicates independently of the main chromosome

• Small - easily taken up by bacteria

• Circular - stable

• Contain origin of replication - can replicate independently and produce large numbers of plasmids

• Restriction sites - genes can be added

• Marker genes (eg antibiotic resistance or GFP) - transformed bacteria can be identified

What are examples of vectors used to move genes into another organism

• plasmids

• Viruses (eg retroviruses)

• Liposomes - tiny spheres of lipid containing DNA

How can bacteria be forced to take up plasmid vectors

by being placed int concentrated calcium ion solution and ‘heat shocked’ - put in very hot environment, then cold, then hot etc. the bacteria become transformed

How can plasmids be inserted into plant cells, and what type of plasmid 

• specific bacterium Agrobacterium tumefaciens

• Ti plasmids 

What are GFPs and what can it be used as

Green fluorescent proteins, as a selectable markers

How do GFPs work as selectable markers

• transformed cells produce the protein which is fluorescent under UV light

• Use a gene which produces an enzyme which converts colourless substrates into colourless ones therefore a small quantity of protein can produce a visible result.

What is the danger of using antibiotic resistant genes as marker genes

Gene could be transferred into other species of potentially pathogenic bacteria

What needs to happen to a gene to produce their protein and what is it controlled by 

needs to be expressed, controlled by the promoter region where RNA polymerase binds. 

What is needed to transfer genes from eukaryotes to prokaryotes

an expression vector (ie plasmid with relevant promoter) so that desired gene is expressed

How was the gene for human insulin expressed in bacteria

• enzyme β-galactosidase only expressed in the presence of lactose

• Insulin gene was inserted next to the β-galactosidase gene so they shared a promoter.

• By growing bacterium in presence of lactose, both genes would be expressed and insulin produced

What does gene editing allow

genetic material to be added, removed or altered at specific locations in the genome, thus offering the treatment of diseases by cutting out or replacing mutated genes

How does gene editing differ from genetic engineering

Gene editing involves the modification of the existing DNA

How does the gene editing technique CRISPR work

• uses short RNA template which matches the DNA target sequence in the genome

• RNA template guides Cas9 enzyme to targeted DNA sequence

• Cas9 cuts genome at this point to allow the editing to occur