4: Genetic Engineering Flashcards
(39 cards)
What is recombinant DNA?
An artificial stretch of DNA assembled in a precise way
Often needs to be in a particular orientation
Built on the backbone of a plasmid
What are the four requirements for recombinant DNA technology?
1) Restriction endonuclease (scissors)
2) DNA ligase (glue)
3)Plasmids (backbone)
4) E.coli (factory to amplify)
What are restriction endonucleases?
Enzymes discovered from bacteria in defence against bacteriophages (viruses that attack bacteria)
Restriction endonucleases “restrict” infection, bacteria use them to bind to and cut short DNA sequences in the bacteriophage genome to prevent replication
What was the first restriction endonuclease discovered?
EcoRI
Cuts the phage genome as it enters the bacterium
Binds DNA as a homodimer in a symmetrical way
Uses magnesium ions to catalyse the cleavage of DNA
Cuts between the G and A of GAATTC on both DNA strands
Causes creation of sticky ends and a 5’ overhang
Why doesn’t EcoRI cut its host DNA?
Because the bacterial genome is methylated
EcoRI therefore cannot cut it
Describe the type of sequence that restriction endonucleases tend to recognise.
Palindromic sequences
(the same forward and backward from 5’-3’ and 3-‘5’)
E.g. GAATTC and CTTAAG
They always cut between the same two nucleotides, breaking the phosphodiester bond and adding a water molecule
What is the difference between sticky ends and blunt ends?
Sticky ends have an overhang, they have a greater surface area for bases to form complementary pairs, therefore stronger ligation occurs
Blunt ends do not form overhangs and therefore are less likely to ligate
What length are restriction endonuclease recognition sites?
4-8bp
Palindromic
What is T4 DNA ligase?
The enzyme that catalyses the formation of phosphodiester bonds in double-stranded DNA molecules
Requires juxtaposed (nearby) 5’ phosphate and 3’ hydroxyl
Requires ATP as a cofactor
Isolated from T4 bacteriophage
Requires sticky ends for best function
Can work on blunt ends but is less efficient
What are the three things a plasmid needs for RDT?
1) Unique cloning sites (e.g. doesn’t exist on any other point on the plasmid)
2) Drug-resistant gene (e.g. antibiotic resistant)
3) Origin of replication (ori)
What is the origin of replication?
The specific sequence of DNA where DNA replication begins, allowing the plasmid to replicate independently within a host cell
Describe how plasmids can be used to build recombinant DNA.
1) Open plasmid using restriction endonuclease
2) Add DNA cut with the same restriction endonuclease so it will be compatible
3) Join via T4 DNA ligase
*often the plasmid
What is the general success rate of RDT with plasmids?
Low
Generally, the plasmid will re-ligate itself before the DNA has time to be incorporated
90% self-ligated plasmids are produced, and 10% recombinant plasmids
How many cloning sites do most plasmids usually have?
10
What is double digest?
Use of two restriction enzymes to cut at two different recognition sites
This ensures more specific fragmentation as it ensures directionality and creates sticky ends for more efficient cloning
What is transformation?
Getting the DNA into the cell
Either using:
1) CaCl2 and heat-shock to increase permeability of the membrane by neutralising the membrane charge
B) Using high voltage electroporation to create temporary pores to allow DNA to pass through
How do you amplify recombinant DNA once successfully produced?
Usually using bacteria like E.coli
Transformed bacterial will replicate when cultured with the bacteria
If recombinant DNA is inserted into a plasmid with antibiotic resistant gene, antibiotic selection can be used to isolate only the bacteria that have successfully taken up the plasmid
What is PCR?
Polymerase chain reaction
Used to amplify small DNA fragments
Describe how PCR is carried out.
1) Denature the DNA
2) Anneal primers to complementary sequences
3) Extension via Taq polymerase
4) Use T4 DNA ligase to ligate blunt ends
5)Can use GFP to insert into vector for expression and detection
What are the temperature changes needed during PCR?
1) Denaturing DNA: 95℃
2) Annealing primers: 50-65℃
3) Taq polymerase: 72℃
What are PCR primers and what are their adaptors?
PCR primers are short, single-stranded sequences that flank target DNA and provide starting points for DNA synthesis
Adaptors are additional sequences that can be added to primers so that the PCR product has desired features at its ends, e.g. enabling ligation to a vector
At what stage of the embryo is genome engineering usually carried out?
In the zygote (fertilised egg)
Because the zygote is the founder cell of the whole organism, so anything done to the genome of the zygote will be inherited by all cells, including the germline
What three things do transgenes need?
1) Gene of interest (e.g. GFP)
2) Ubiquitous transcriptional promoter
3) Polyadenylation signal
What is a ubiquitous transcriptional promoter?
A DNA sequence that drives expression of the downstream gene
In contrast to a specific tissue promoter
The promoter brings in RNA polymerase machinery to start transcription
