DNA Technology Flashcards
What produces the reverse transcriptase enzyme
Retroviruses
Reverse transcriptase creates a DNA fragment from what
MRNA
What are the stages to reverse transcriptase
Select a cell that readily produces the desired protein
Extract mRNA from the cell for the desired protein
Use reverse transcriptase to synthesise complementary DNA strand
Used DNA polymerase too add complimentary nucleotides on the DNA template
What produces restriction endonucleases
Bactria
What are the two types of cuts that restriction endonucleases make
Blunt ends
Sticky ends
What are the stages to using a restriction endonuclease
Specific enzyme chosen for it specific cutting point
Enzyme finds recognition sequence and makes cut to form sticky ends
DNA and plasmid from Bactria can be cut at same point using the same restriction endonuclease
What is DAN ligase’s role
Join together DNA fragments with complimentary sticky ends
Completes sugar phosphate backbone so a stable double strand is formed
Means they can be used to piece together different DNA fragments
What is recombinant DNA
A DNA strand formed from to different sources
What are the two ways to produce the DNA fragments
Restriction endonuclease
Reverse transcriptase
Explain bacterial transformation
Use recombination plasmid and insert it into bacteria
Add calcium ions and heat
Bacteria becomes permeable
What are the problems with bacterial transformation
Very few Bactria take up the plasmid
Recombinant plasmid dies not always form
How do you identify Bactria that have successfully taken up a plasmid using antibiotic resistance
Find plasmid with resistance to two antibiotics
Inserted desired gene in the middle of one antibiotic resistance gene using restriction endonuclease
Grow the Bactria on agar jelly plate
Create a copy but on a plate with the antibiotic that’s resistance gene has not been interfered with
Then do the same again but with the antibiotic that the resistance gene has been interfered with
The colones on the plate with the uninterrupted antibiotic plate that are not on the disrupted plate are the cosines that have successfully taken up the plasmid
Explain how fluorescent markers can be used to indicate a vector has up taken a desired plasmid
Insert gene for GFP
Desired gene inserted into GFP green
Bactria which do not fluoresce have taken up the plasmid
Explain how enzyme markers can be used to indicate which vectors have taken up the desired plasmid
Insert gene for lactase
Lactase will turn a colourless substrate blue
Desired gene inserted into lactase gene
Bactria which do not turn substrate blue will have taken up plasmid
