genetic engineering and ethics Flashcards
what is recombinant DNA and what is used for it
combining DNA from different organisms/ species
-genes isolated from one organism and inserted to another using vector
what is the tool kit for genetic engineering
-reverse transcriptase= mRNA turned into DNA
-restriction enzymes
-ligase enzymes= join dna together
-vectors
-markers
-probes
explain how restriction enzymes work
-from bacteria in which are used to protect against attack from phage viruses= cut viral DNA preventing virus from replicating= prokaryotic DNA is protected by being methylated at recognition site
-used in manipulating genomes as molecular scissiors
-make staggered cut= sticky ends
explain how ligase enzymes work
used to join DNA fragments catalysing condensation reaction joining sugar groups and phosphate groups of DNA (sugar phosphate backbone)
-present in DNA replication and pcr
how does reverse transcritase work
opposite of transcription= makes DNA from RNA
explain the 5 stages of genetic engineering
- isolation= obtaining required gene
- insertion= placing copy of gene inside vector
- transformation= vector carries the gene into a recipient cell
- identification= which cells are transformed
- recipient cell expresses the gene
explain the option of finding a template for the isolation stage of obtaining a gene
-obtain mRNA from cells expressing desired gene
-reverse transcriptase enzyme produces single strand of complementary DNA -cDNA- using mRNA as template
-add primers and DNA polymerase will make the cDNA into double stranded length of DNA
explain the option of gene making machine for the isolation stage of obtaining a gene
if nucleotide sequence of the gene is known then the gene can be synthesised using an automated polynucleotide synthesiser
explain the option of PCR for the isolation stage of obtaining a gene
PCR primers can be designed to amplify genes from genomic DNA if the sequence of the gene is known= can find dna
explain the option of cutting out genes using restriction endonucleases for the isolation stage of obtaining a gene
-use of DNA probe to locate the desired gene within genome
-use restriction enzymes to cut gene out
what are restriction endonucleases, recognition site, sticky ends
-enzymes that cut DNA at specific recognition site
-site enzyme ats on breaking hydrogen bonds
-short pieces of single strand at the end of cut dna
how is the gene inserted in a vector
-plasmid taken from bacteria are mixed with restriction enzymes that cut plasmid at specific recognition sites leaving sticky ends
-nucleotide bases added complimentary to sticky ends of plasmid, to the gene to be inserted = easily join by hydrogen bonds
-gene and plasmid anneal using DNA ligase
why is the same restriction enzyme used to cut open the plasmid vector
to end up with complimentary sticky ends
what are the options for transformation (carrying vector into recipient cell
1- heat shock treatment= alternating hot and cold temps with calcium chloride present= membrane more porous
2-electroporation= electricity across membrane= disrupts= plasmid enters
3- electrofusion
4- transfection= DNA packaged in a bacteriophage (virus infecting bacteria)= transfect the host cells
5- A. tumefaciens in plants (bacteria infecting plants)
what can be used if plant isn’t susceptible to A. tumefaciens
gene gun, small pieces of gold or tungsten can be coated with DNA and shot into plant
what is a BAC
bacterial artificial chromosome
explain how insulin is made from GM bacteria
-isolation of human gene responsible for insulin production (mRNA converted into cDNA then DNA)
-plasmid is cut out with restriction enzymes
-DNA inserted into plasmid and sealed using DNA ligase
-plasmid with gene inserted into bacterium (one of transformation techniques)
-reproduction of bacteria and plasmids= insulin produced
what is identification of transformed for, what is a genetic marker and examples?
-used to identify host cells that have successfully taken up gene of interest by the use of genetic markers e.g
-antibiotic resistance genes
-fluorescent markers
-enzyme markers
