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

Manufacture

Recombinant plasmid introduced to bacteria

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

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