genetic engineering Flashcards
what is recombinant DNA
when DNA from an organism is formed from different organisms
what is meant by the term transgenic
the name for organisms which contain DNA from another organism
what enzymes are needed for genetic engineering to occur
1) Restriction endonucleases
2) DNA ligase- joins sugar phosphate backbones of DNA sections
3) reverse transcriptase
4) DNA polymerase - joins single stranded DNA to form a double stranded DNA
what do restriction endonucleases do
they cut DNA at short, specific and palindromic sequences at the restriction sites producing a staggered cut of unpaired bases called sticky ends
each restriction enzyme is specific to one restriction site eg one enzyme will only cut ATTC whilst the other will only cut TTAA
what is reverse transcriptase
an enzyme which synthesises single stranded DNA from an mRNA template
what are the different ways of obtaining the desired gene from a DNA strand
1) gene can be synthesized by an automated polynucleotide sequencer= only if desired gene sequence is already known
2) The use of gel electrophoresis and a DNA probe
3) the synthesis of cDNA
outline how the use of a DNA probe can be used to obtain the desired gene
1) DNA strand is fragmented using restriction endonucleases producing sticky ends eg AATT
2) the fragments are then separated using gel electrophoresis
3) A DNA probe is a single stranded piece of DNA complementary to the desired gene in this case TTAA
4) DNA probe is usually marked so when binds to complementary DNA fragment of desired gene it is easy to spot
outline how the desired gene can be obtained using mRNA
1) Isolate the mRNA of the desired gene from cells actively synthesising it
2) convert the single stranded mRNA into a single stranded DNA using reverse transcriptase
3) Use DNA polymerase to form a double stranded cDNA (copy DNA)
outline how recombinant DNA is formed using a vectors plasmids
1) cells containing the plasmids are treated with chemicals dissolving the cell walls
2) ultracentrifugation is used to separate plasmids from the rest of the cell debris
3) Use the SAME restriction enzyme, used to obtain the desired gene, to cut the same gene producing complementary sticky ends to the desired gene
4) use DNA ligase to join the donor DNA with the vector DNA which joins the sugar-phosphate backbone forming recombinant DNA
what problems can you face when mixing the cut plasmid, DNA ligase and the isolated genes
either the DNA ligase will combine the plasmids with the isolated gene which is wanted or
the DNA ligase will just reseal the original plasmid back up
what method is used to ensure plasmids are taken back into the bacteria
1) mix plasmids with the bacteria cell
2) use heat shocking, chilling to 4 degrees then heating to 42 degrees, and the addition of Ca+
3) this causes a small number of the plasmids to be taken up- 1%
4) bacteria are now transformed as they have taken DNA from outside the cell
what is the issue face when the plasmids have been taken into the bacteria
three different types will be formed
1) bacteria that are not transformed= take up nothing
2) bacteria that have taken up plasmid with no donor DNA as they were resealed by DNA ligase
3) bacteria taking up the recombinant DNA= WANTED
outline how genetic markers are used to distinguish between recombinant bacteria and non.
1) plasmids used in the beginning are coded with 2 genes showing antibiotic resistance to ampicillin and tetracycline
2) when plasmids are cut using a specific restriction enzyme, whose restriction site is the middle of the tetracycline gene.
3) if the wanted gene is inserted into the plasmid the resistant gene will no longer work showing recombinant DNA
4) but if DNA ligase resealed the original plasmid the resistant gene will still work
5) replica plating is used to tell the 3 different types of bacteria apart
outline replica plating
1) the colonies of bacteria are grown on a master plate and transferred onto an agar plate containing ampicillin
2) bacteria that took up no plasmids at all will have no plasmids with the antibiotic-resistant gene, so will die, leaving with the recombinant bacteria or the resealed plasmid bacteria
3) the surviving bacteria are transferred to another plate containing tetracycline
4) bacteria with the resealed plasmids will survive as the resistant gene is still intact whilst the ones with the recombinant DNA, which we want, will die
5) return to the master plate and collect the corresponding bacterial colonies and scale-up
what are the advantages of recombinant DNA
1) no limit to the amount of protein that can be synthesised
2) allows the synthesis of complicated protein structures which can only be synthesized by living cells
