3.5 Genetic modification and biotechnology Flashcards
What does PCR stand for?
Polymerase chain reaction
What is PCR?
An artificial method of replicating DNA in lab conditions
What is the PCR technique used to do?
Amplify large quantities of a specific sequence of DNA from an initial minute sample
What does each cycle of PCR do to the amount of DNA?
Double it
Where does PCR occur?
In a thermal cycler
What does PCR use to control the replication process?
Variations in temperature
What are the three stages of PCR?
Denaturation
Annealing
Elongation
What happens during the denaturation stage of PCR?
DNA sample is heated to separate it into two single strands
What is the temperature for the denaturation stage of PCR?
95C for 1 min
What happens during the annealing stage of PCR?
DNA primers attach to the 3’ ends of the target sequence
What is the temperature during the annealing stage of PCR?
55C for 1 min
What happens during the elongation stage of PCR?
A heat tolerant DNA polymerase binds to the primer and copies the strand
What is the temperature used during the elongation stage of PCR?
72C for 2 min
After PCR and once large quantities of DNA have been created what happens?
Other lab techniques are used to isolate and manipulate the sequences
What is gel electrophoresis?
A lab technique used to separate and isolate proteins or DNA fragments based on size
What happens in gel electrophoresis?
Samples are placed in a block of gel and an electric current is applied which causes the samples to move through the gel
Why will smaller samples move faster through the gel in gel electrophoresis?
As they are less impeded by the gel matrix
Why do samples of different sizes separate?
As they travel at different speeds
How may DNA be cut into fragments?
Using restriction endonuclease
What will different DNA samples generate?
Different fragment lengths
In DNA separation, why do fragments separate?
Because DNA is negatively charged due to the presence of a phosphate group on each nucleotide
In DNA separation, where are DNA samples placed?
Into an agarose gel
In DNA separation, how is fragment size calculated?
By comparing against known industry standards
In DNA separation how can specific sequences be indentified?
By incorporating a complementary radiolabelled hybridisation probe, transferring the separated sequences to a membrane and then visualising via autoradiography
In protein separation, what must proteins first be treated with?
An anionic detergent
In protein separation,why must proteins be treated with an anionic detergent?
To linearise and impart a uniform negative charge
In protein separation, where are protein samples placed?
In polyacrylamide gel
In protein separation, what are sizes compared against?
Known industry standards
In protein separation, where are separated proteins transferred to?
A membrane
In protein separation, how are target proteins identified?
By staining with specific monoclonal antibodies
How can proteins affect size?
They may be folded into a variety of shapes
How do proteins have no clear charge?
They have positive and negative regions
