8 The Control of Gene Expression- Gene Technology Flashcards
What is recombinant DNA and how is it produced?
-Where fragments of DNA are inserted into other sections of DNA
-Genetic code= universal; any section of DNA can be taken from one organism, placed inside another
-It is then transcribed & translated to produce proteins (also universal processes)
-Process of transferring sections of DNA produces recombinant DNA
What are fragments and transgenic organisms?
-The sections of DNA that are transferred
-Organism that has received fragments of DNA
Why are DNA fragments produced and what are the ways in which this happens?
-Recombinant DNA often involves inserting specific gene of interest into DNA; gene normally encodes protein w/ useful properties. Transferred gene= target gene
-To transfer target gene, it needs to be removed from DNA in section called a DNA fragment, can be produced in three ways:
-Reverse transcriptase
-Restriction endonuclease
-Using a gene machine
What can be done to DNA fragments after they have been produced?
They can be amplified either in vivo (inside organism) or in vitro (outside of organism)
What happens after a fragment has been produced?
It can be inserted into the genome to produce a transgenic organism
How can reverse transcription be used to produce DNA fragments?
-mRNA; single-stranded molecule produced when a specific length of DNA (target gene) is transcribed, it is complementary to base sequence in target gene
-mRNA sequence can be used as template for producing fragments of DNA
-Reverse transcriptase= enzyme that converts single-stranded mRNA into double-stranded DNA, can be used in producing DNA fragments by converting mRNA for target gene into double-stranded DNA
-DNA produced= complementary DNA (cDNA)
How are DNA fragments of insulin isolated from pancreatic cells?
-mRNA for insulin → isolated from pancreatic cells
-mRNA is mixed w/ reverse transcriptase
-This converts mRNA → cDNA
-cDNA can be used to produce recombinant DNA
What are recognition sequences and their function?
-Sections of DNA where base sequence has palindromic base pairs (have sequence of base pairs that are the same but in opposite directions)
-Can be used to isolate target gene if there are 2 sets of sequences either side of gene
How are DNA fragments produced using restriction endonucleases?
-DNA containing target gene is mixed w/ restriction endonuclease enzymes
-Restriction endonucleases bind to recognition sequences on either side of target gene
-Target gene is cut out of DNA
How can DNA fragments be produced using gene machines?
-DNA fragments can be produced by synthesising target gene sequence w/ free-floating nucleotides; useful as DNA template isn’t necessary
-Sequence for target gene—> obtained from database
-Nucleotides are added in correct order to synthesise correct base sequence
-Protecting groups added throughout synthesis to make sure correct nucleotides are added & no side branches are produced
What are the steps involved for in vivo amplification?
-Vector= form of transport for DNA fragment. Vector DNA is cut open at specific regions (recognition sequences) by restriction endonuclease enzymes so each end has a short single-stranded section; the sticky ends
-DNA fragments have sticky ends complementary to sticky ends on vector DNA as they’ve either been cut from DNA using same restriction endonucleases/several nucleotides have been added onto ends of fragment.
-They bind together as DNA ligase enzyme attaches sticky ends together; this is called ligation
-DNA fragment has been inserted into vector DNA; this is recombinant DNA
-Vector transfers recombinant DNA to host cell. If it’s a plasmid: host cells take up recombinant DNA via heat-shock (where cells are heated at 42°C for 1m). If it’s a bacteriophage: recombinant DNA injected into host cells
-Cells successfully taken up the recombinant DNA are transformed; said to be genetically modified but not all will be (transformed cells identified using marker genes- these are inserted along w/ recombinant DNA & confer antibiotic resistance
-Transformed cells can be identified by placing them on agar plate w/ antibiotics; only cells that have successfully taken up recombinant DNA will be able to survive
-Transformed cells can be grown in large # to amplify target gene
What are the steps involved for in vitro amplification?
-DNA fragments are mixed w/; primers (short sections of DNA), enzyme DNA polymerase (produces new strands of DNA), free-floating nucleotides
-Together these components form reaction mixture
-Heat reaction mixture to 95°C; high heat causes hydrogen bonds between DNA strands to break & DNA to separate into 2 separate strands
-Cool reaction mixture to 65°C; causes primer to anneal (recombine) to the 2 separate strands of DNA. Primers= complementary to beginning of 2 strands
-Heat reaction mixture to 72°C (optimum temperature for DNA polymerase activity)
-DNA polymerase produces 2 new DNA strands by using the two separated strands of DNA as template & adds free-floating nucleotides complementary to template strands of DNA
-Primers allow nucleotides to bind to one another, produce strand of DNA
-This process of heating, cooling & heating produces 2 new strands of DNA from 1
-Can be repeated as many times as possible to quickly amplify # of DNA fragments; doubled in each cycle of PCR
How is recombinant DNA used for genetically modified crops and why is this beneficial?
-Can be used to genetically modify crops to improve their yield
-Traits that can be improved; resistance to disease, tolerance to application of herbicides & pesticides, tolerance of adverse environmental conditions
How can recombinant DNA be used for genetically modified livestock and why is this beneficial?
-Can be used by farmers to make production of meat more economically viable
-Traits that can be improved; grow faster & larger, resistance to disease
How can recombinant DNA be used in nutrition?
Can be used to increase nutritional value of food
How can recombinant DNA be used in treating diseases?
Can be used to produce medicine and hormones to treat diseases
How can recombinant DNA be used to benefit industry?
Can be used to manufacture enzymes, which can then be used in industry
What are the ethical concerns to do with the spread of genes from recombinant DNA?
-GM crops & livestock → produced when beneficial gene is inserted into their genome to improve certain trait
-Genes could be transferred into other organisms where it’s harmful
-Genes from genetically engineered (transgenic) crops could also be spread to organic crops
What are the ethical concerns to do with the unforeseen impacts of recombinant DNA?
-Genetic modifications to an organism could have unforeseen effects & disrupt normal gene function
-Use of genetically engineered organisms could lead to ↓ in variety in populations
What are the ethical concerns to do with the economic consequences from recombinant DNA?
-Could be economic consequences for some countries if genetically engineered crops can be grown in different countries where it wasn’t previously possible
-Companies that are able to invest more money in recombinant DNA technology may out-compete others
What are the ethical concerns to do with medical uses of recombinant DNA?
Some activists are concerned that using recombinant DNA in medicine could lead to unethical uses of genetic engineering
What is gene therapy?
A genetic engineering technique used to cure disease
What is the procedure of gene therapy?
-Introduction of target gene into genome
-It’s then transcribed & translated to produce desired protein
-Protein counteracts effect of disease caused by a mutation
How does gene therapy vary based on allele interactions?
-The way that gene therapy is used depends on allele interactions of gene causing disease
-If mutation is in recessive allele; wild-type (typical of species) dominant allele inserted into genome, counteracts mutant alleles
-If mutation is in dominant allele; allele that ‘silences’ mutant allele is inserted in genome
