Rod Devenish; Week 5 MED1011; Biochemistry
Restriction enzymes are made by microbes as a defence against viruses or foreign DNA, binds to DNA and cuts it at specific sequences. Methyl groups at restriction sites block restriction enzymes and stop cleavage in the host genome. Hybridisation of flourescent tags can be used to identify fragments. Fragments from restriction enzymes can be separated by size by gel electrophoresis, slices of gel can be cut out and the DNA fragment purified for use. Shorter fragments are moved further towards the positive end of the plate, longer stuck at negative end. DNA probes can be added to this gel electrophoresis and bind to target strands. Many restriction enzymes make staggered cuts in DNA, creating sticky ends with unpaired bases. This end can be used to make recombinant DNA if DNA from the same molecules is made with the same restriction enzymes. Different parts can be sealed with DNA ligase. This can be utilised to have dual resistance to antibiotics by modifying plasmids. Newly introduced DNA must be part of a DNA carrier molecule, or vector. Bacteria, yeasts and cultured plant cells are commonly used.
Vectors must contain a replication unit, recognition sequences for restriction enzymes so DNA can be inserted, and genetic (reporter) markers to identify their presence in host cells. Vectors are introduced through biological, chemical or mechanical means: virus, chemical treatment (high Ca), liposomes, electroporation, microinjection, bombardment with DNA coated particles. Nutritional, antibiotic resistance or flourescent markers can identify what cells contain the vector. Cutting of DNA by restriction enzymes can allow the fragments to be combined with a vector and inserted into a host to create a gene library (genomic DNA fragments inserted into plasmids and then bacteria).
mRNA produced at a certain time can be harvested and used to produce complimentary DNA (cDNA), which can then be cloned like gene fragments to create a library. It represents only the genes expressed when mRNA was obtained. cDNA can create more cDNA.
Another source of DNA is synthetic DNA. By making mutated genes the effect on protein and function can be measured. Transgenes can be injected into eggs and cultured, transferred to foster mothers that may incorporate transgene into germ line. Transgenic animals tested with PCR or hybridisation (Southern blotting). Transgenes can mate and produce homozygous for transgene. Homologous recombination can be used to 'knock out' a gene- this is done by cloning DNA of interest into a vector and adding DNA to disable it. Mouse embryonic cells are transfected with DNA and homologous recombination knocks out the normal functioning copy (rearranging chromosomes in meiosis). Transfected cells are screened for the desired modification and then are injected into a mouse to hopefully end up in germ line. Homozygous knockout mice can occur through mating. Can be used as disease models.
Antisense RNA can be generated to be complimentary to mRNA, bind and inactivate it to downregulate gene expression. Hybrids are broken down rapidly. siRNA (small interfering) can also be used to block mRNA, is double stranded unwound by a protein complex, guides siRNA to complementary mRNA and catalyses its breakdown.
PCR is used to repeatedly replicate DNA. Double stranded DNA is heated. Short (15-20 bases) of very specific and chemically synthesised primer strands are added specific for the 3' end of complementary strands. Heat stable DNA polymerase is added. Creates a geometric increase.