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19.2 Tools for the gene technologist - Coggle Diagram
19.2 Tools for the gene technologist
Restriction enzymes
Made by bacteria to cut up phage DNA and restrict infection
Makes cuts at specific target sites along sugar-phosphate backbone within the DNA molecule
Gene probe
Single stranded DNA with known sequence complementary to target sequence
Binds to target DNA and allows detection of specific sequences
Reverse transcriptase
Copying eukaryotic DNA for use in prokaryotes does not work, as eukaryotic genes have exons (coding) and introns (non-coding), which bacteria do not have and cannot deal with
mRNA is taken from the cytoplasm (so introns have already been removed)
mRNA is converted into single-stranded DNA via reverse transcriptase
Single-stranded DNA is converted into double-stranded DNA using DNA polymerase
Made by RNA viruses
Synthesising DNA
Use of genetic code to make new genes by choosing codons for the desired amino acid sequence
DNA synthesiser machines make short DNA fragments and then join them together
Plasmids as vectors
Obtaining
Treat bacteria with enzymes to break down cell walls
Centrifuge to separate large circular DNA from plasmids
Recombining
Use same restriction enzyme to cut open plasmids and also isolate target gene to form complementary sticky ends
Isolated gene and cut-open plasmids are mixed
Sticky ends of gene may pair up with sticky ends of plasmid
Plasmid may simply reseal without incorporating gene
DNA ligase forms sugar-phosphate backbone by catalysing formation of phosphodiester bonds
Features of good plasmid vector
Low molecular mass so readily taken up by bacteria
Origin of replication so easily copied
Several single target sites for different restriction enzymes (found in short length of DNA called polylinker)
Marker genes so able to identify cells that have taken up plasmid
Insertion into bacteria
Mix bacteria in high conc. Ca2+ solution
Cool and then heat (heat shock)
Recombinant human insulin
Isolation of human gene and preparation of vector
mRNA for insulin taken from pancreatic beta cells
mRNA converted to ssDNA via reverse transcriptase
ssDNA converted to dsDNA via DNA polymerase
Restriction enzyme used to cut plasmid and allow dsDNA to combine
DNA ligase used to produce recombinant plasmid
Manufacture
Recombinant plasmid introduced to bacteria
Transformed bacteria identified and grown
Bacteria transferred to industrial fermenters
Genetic markers
Green fluorescent protein (GFP) causes glowing under UV light
Beta-glucuronidase (GUS) causes colour formation
Promoters
A length of DNA to which RNA polymerase binds to at start of transcription