Gene technology Flashcards
How can DNA fragments be made?
Conversion of mRNA to DNA using reverse transcriptase (cDNA)
Cut out DNA section using restriction endonucleases
Create a gene in a gene machine
What does in vitro mean?
Outside a living organism
In glassware
What does in vivo mean?
Carried out inside a living organism
For what is PCR an abbreviation?
Polymerase chain reaction
List the items needed to perform PCR
Thermocycler
Primers
DNA Nucleotides
DNA polymerase
Section of DNA to copy
In PCR the thermocycler initially heats the contents to 95 oC. What happens at this temperature?
DNA double helix separates into single strands
Hydrogen bonds between complementary base pairs are broken
What is a primer?
A short piece of DNA (15-20 bases long) with complementary base sequence to the ends of the DNA fragment you wish to copy
At what temperature do primers bind to the DNA fragments?
55oC
What is taq DNA polymerase?
DNA polymerase that is not denatured at high temperatures.
Where is taq DNA obtained from?
Thermophilic bacteria which live in hot springs
eg Thermus aquaticus
Why is a thermocycler used in PCR?
A computer controlled machine which allows temperatures to be controlled and changed quickly and accurately
Cycle take about 2 minutes
What is the function of the DNA polymerase in a PCR machine
Catalyses the formation of phosphodiester bonds between nucleotides
What is the advantage of using in vitro cloning?
Quick
No living cells needed
Give the sequence of temperatures used in PCR and state what happens at each temperature
95oC - breaks hydrogen bonds between DNA strands
55oC - primers anneal to DNA strands
72oC - DNA polymerase catalyses the formation of phosphodiester bonds between nucleotides
