BGM1004/L05 Recombinant DNA Technology I Flashcards
Give the 3 principles of recombinant DNA technology.
Creation of recombinant DNA
Cloning of recombinant DNA
Using recombinant DNA
Describe creation of recombinant DNA. (2)
Construction of new combinations of unrelated genes
2 pieces ligated to perform modified function
Describe cloning of recombinant DNA.
Amplifying new DNA many times to introduce into living cells
Describe using recombinant DNA.
Expressing a cloned gene to produce a protein
Give an overview of how to create recombinant DNA. (3)
Take 2 pieces of DNA from unrelated sources
Use biological environment to clone and amplify
Express cloned DNA to make a protein
Give 4 types of host cell.
Yeast
Bacteria
Mammalian cells
Human cells
Give an advantage of using yeast and bacterial cells as host cells.
Easy to manipulate
Give an advantage of yeast cells over bacterial cells as a host.
Yeast cells eukaryotic so closer to mammalian cells
Give a disadvantage to human and mammalian cells as a host.
Difficult to manipulate as they easily become infected
Name the 4 enzymes involved in recombinant DNA synthesis.
Restriction enzymes
DNA ligase
Taq polymerase
Reverse transcriptase
What do restriction enzymes do?
Cleave DNA at specific sequences
What does DNA ligase do?
Ligates fragments of DNA together through phosphate backbone
What does Taq polymerase do?
Copies DNA many times
What does reverse transcriptase do?
Copies RNA into DNA
Give 2 types of cleavage.
Symmetrical
Asymmetrical
What enzyme can ligate 2 pieces of DNA cut by the same restriction enzyme?
DNA ligase
What 2 types of DNA are required to create recombinant DNA?
Vector DNA
Insert DNA
What 5 features must recombinant DNA have?
Unique restriction site
Efficient oriC
Selective gene
Regulatory sequences
Easily introduced into host
How long are plasmids?
2-200kbp
What is a polylinker?
Insert of DNA with recognition sites for many restriction enzymes
Give 2 other examples of vectors.
Bacteriophages
Cosmids
Phagemids
Give 2 uses of recombinant DNA.
Expression
Recovery and further manipulation
What process is useful for amplifying a specific piece of DNA?
Polymerase Chain Reaction (PCR)
How are cells made competent? (2)
Heat shocking and adding CaCl
What temperature are cells incubated at for recombinant DNA transformation?
37C
How long are cells incubated for recombinant DNA transformation?
30 mins
How can transformed cells be selectively cultured?
Grown on a selective medium e.g., containing antibiotic
