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Pack 15 - Recombinant DNA technology - Coggle Diagram
Pack 15 - Recombinant DNA technology
Definitions
Recombinant DNA
The combination of DNA together from two different organisms
Transgenic organism
An organism that had had its genome modified
Genetically Modified Organism (GMOs)
In Vivo replication
Process takes place in living organism (host cell) using vector
Isolation
Insertion
Transformation
Identification
Growth/Cloning
Host cells containing the DNA fragment can now be cultured on an industrial scale to produce large quantities of the required protein, or the genes themselves used for gene therapy
Of host cells that have taken up the gene using gene markers
Antibiotic resistance, Fluorescence or Enzyme(LacZ) marker gene attached to gene of interest to identify it
Transfer gene and vector into suitable host cells eg E. coli
Bacterial cells mixed with calcium ions to make them permeable and then chilled on ice, then warmed to 42 degrees (heat shock/pulse). Or electroporation
Of gene into a vector (a carrier) eg plasmid
Restriction endonuclease
group of enzymes that cut DNA at specific recognition sequences. Can cut to produce staggered ("sticky") ends, or blunt ends. - recognition sequences are often 6 base pairs in length and palindromic
Add promoter + terminator region to the fragment of DNA to allow for RNA polymerase to bind and disengage and production of mRNA.
Cut both target gene and vector(DNA molecules used to carry foreign DNA into cell to be replicated or expressed) with the same restriction endonuclease - mix together so H-bonds form between complementary strands. Add DNA ligase to connect sugar phosphate backbone on both strands (ligation)
Production of DNA fragments that have the required gene
using mature mRNA(pre-mRNA with introns removed) reverse transcriptase can be used to make complementary DNA (cDNA) to allow the strand to act as a template to produce double stranded DNA of the target gene without introns
In Vitro Replication
Process takes place "in glass" - not in living organism, Polymerase Chain Reaction (PCR)
Polymerase Chain Reaction (PCR)
Gene Machine
If sequence of a gene is known it can be artificially made:
Nucleotide sequence put into computer and checked for biosafety
Computer designs series of small, overlapping, single stranded DNA (oligonucleotides)
Oligonucleotides joined to make whole gene (no introns)
Gene replicated via PCR then inserted into bacterial plasmid
Genes sequences and errors rejected
Correct genes can be transferred to host cell for replication