3.8.4.1 - in vivo and in vitro gene cloning

what is recombinant DNA

when DNA from 2 different organisms has been combined, resulting in a transgenic organism (genetically modified organism/GMO)

what is in vivo gene cloning

inserting DNA into a living host cell for replication and production of a useful product (protein)

advantages of in vivo gene cloning

useful to introduce gene into another organism, involves almost no risk of contamination, very accurate, cuts out specific genes, produces transformed bacteria that can be used to produce large quantities of gene products e.g. proteins

5 key stages of making recomnbinant DNA in vivo

isolation of DNA fragments containing gene coding for desired protein, insertion of gene into vector e.g. plasmid, transformation/transfer of DNA into suitable host cells, identification of host cells that successfully incorporated new gene into their cells, growth/cloning of cell identified in step 4

what are the 3 ways in which isolation of DNA fragments containing gene coding for desired protein can be done (first stage of in vivo cloning)

converting mRNA to cDNA using reverse transcriptase, using restriction endonucleases specific to base sequence desired, working backwards from the protein to create artificial version of gene using ‘gene machine’

role of reverse transcriptase to convert mRNA to cDNA for isolation of DNA fragments (first stage of in vivo cloning)

cDNA = complementary DNA/ DNA strand produced from RNA using reverse transcriptase, strand can be copied into multiple double stranded DNA genes using DNA polymerase

role of restriction endonucleases (enzymes that break apart polynucleotide DNA strand) in isolation of DNA fragments (first stage of in vivo cloning)

each re cuts DNA strand at specific base sequence (specificity can isolate individual genes), re can cut either straight down middle of double strand to produce 2 blunt ends or in zig-zag staggered fashion so it produces 2 sticky ends that are palindromes of each other

role of gene machines in isolation of DNA fragments (first stage of in vivo cloning)

desired gene nucleotide base sequence determined from desired protein by determining aa sequence, mRNA codons looked up and comp DNA triplets worked out, computer designs oligonucleotides (small, overlapping single nucleotide strands) which can assemble into desired gene in right order, oligonucleotides join to make gene with no introns and is replicated using pcr, pcr constructs double stranded gene and replicates to make many copies, gene can be inserted into vector using sticky ends, genes checked for errors

strengths of using gene machines for gene isolation

any sequence of nucleotides can be produced in short time, high accuracy, artificial genes free of introns so can be transcribed/translated in prokaryotes

how is gene inserted into a vector (second stage of in vivo cloning)

promotor/terminator sequence also inserted into DNA fragment before insertion into vector’s DNA, sticky ends used by using same re on vector dna as og gene so same palindromic sequence sticking out on both, dna ligase connects 2 strands at sticky ends, in most cases vector is a plasmid of a bacteria cell that codes for antibiotic resistance

how is gene transferred/transformed into suitable host cells (third stage of in vivo cloning)

bacterial cell receives dna bathed in solution ca2+ ions and heated, process makes plasma membrane permeable to plasmid so can enter cytoplasm, low success rate as: <1% bacterial cells will successfully uptake plasmid, some plasmids reclosed as did not bind with target dna, sometimes dna fragment forms its own mini plasmid

how are the host cells that successfully incorporates the target dna identified (fourth stage of in vivo cloning)

place bacteria on growth medium containing antibiotic they should be resistant to if transformation successful (cells where dna not succesfully transferred will die), marker genes - can kill non-resistant ones with antibiotics (needed for replica plating), can be fluorescent/easily seen protein, can produce enzyme that is identifiable when substrate applied

what is replica plating in marker genes for identification

problem that by the time you know the target gene is present as the TET gene is inactivated, you have killed the bacteria with TET so a replica is made where 1 copy is killed and other is collected, only let colonies that have been killed grow

how is in vitro gene cloning carried out

polymerase chain reaction (pcr), done electronically so rapid and efficient, higher risk of contamination than in vivo, used to make billions of copies of dna in relatively short time, useful in forensics

ingredients/requirements for pcr

dna fragments with blunt ends, dna polymerase from thermophilic archaea taq polymerase, primers (short chain/20-30 nucleotides with bases complementary to ends of dna fragment, nucleotides, thermocycler (thermostatic machine)

3 stages of pcr

separation/denaturing of dna strand, addition of primers (annealing), synthesis of dna (extension)

how is the dna strand separated/ denatured (first stage of pcr in vitro cloning)

place dna fragments, primers and taq polymerase in thermocycler, increase temperature to 95c, high temp breaks h bonds and separates the 2 strands

how are primers added/ how does annealing occur (second stage of pcr in vitro cloning)

cool mixture to 55c, causes primers to anneal (join) to complementary bases on ends of strands, prevents 2 dna strands from reforming h bonds and rejoining at this temp, allows somewhere for dna polymerase to attach (act as promoter), positions of each primer 5’ to 3’

how is dna synthesised/ extended (third stage of pcr in vitro cloning)

increase temp to 72c (optimum temp for thermophilic dna polymerase), dna polymerase attaches to both primers and works inwards, binding more free nucleotides complementary to the dna fragment, 2 new dna fragments produced