R.O Lecture 3 Flashcards
Describe Cloning Vectors
Cloning vectors can be used to generate copies of a section of DNA
within a cell.
Specific sections of DNA can be amplified from source material (e.g.
gDNA) and then inserted into a vector.
Cloning vectors contain features that allow the DNA fragment to be
inserted at a specific site.
Why do we use cloning vectors if we can amplify specific DNA by PCR?
-PCR error rates
-Stability of DNA product (storing DNA fragment)
-Off-target PCR amplification (primers can anneal to other regions)
What is the standard cloning vector?
Bacterial Plasmid <3 to 10kb
What is the Plasmid? What does it contain?
- Plamids (small loops of DNA) that can be transmitted from one cell to another
-Plasmids contain ‘non essential’ genes and can be lost or taken up from the environment
-Plasmids can replicate within cells so multiple copies can be found within the same cell
To be useful as cloning vectors, what must plasmids have?
-Multiple cloning site (MCS ) (DNA to be amplified is targeted
to this region of the vector)
-An origin of replication (Ori)
-A selectable marker: This will allow selection of cells that contain the plasmid. Example: tetracylcine resistance gene (tetr)
Where is the DNA of interest inserted?
Multiple Cloning Site (MCS, also knows as polylinker)
Describe Multiple Cloning Site
*Sequence of DNA containing defined sequences of nucleotides
(restriction
*This region is where your ‘DNA of interest’ will be inserted during cloning
*Use of restriction sites in MCS enables targeting of DNA insert to a
specific region on the vector (so as not to inactivate other features of the vector).
*These sites can be specifically cut using restriction enzymes
Describe the role of restriction enzymes
*Restriction enzymes recognise and cut DNA at a specific nucleotide sequence called a restriction site
*Restriction enzymes are isolated from bacteria (defense mechanism)
How can restriction sites and enzymes be used in biotechnology
*Restriction sites and enzymes can be used in biotechnology, research etc. to isolate sections of DNA and target them to a specific region
Restriction enzymes can cut the dsDNA to generate 2 types of ends.
- Blunt ends: Cut at the same point on both DNA strands
- Sticky ends: Cut at staggered points on each DNA strand leaving an overhang
Cuts generally occur at a palindromic sequence (so they read the same in 5’ 5’–> 3’ direction on both strands).
How are restriction enzymes named?
Restriction enzymes are named after the bacteria they were first isolated from (Species and sometimes strain).
example:
EcoRI Escherichia coli strain R, 1st enzyme
EcoN1 Escherichia coli strain N, 1st enzyme
How to introduce your “DNA of interest” into a cloning/expression plasmid.
1.Choose restriction sites present in the MCS of plasmid that are not found in your ‘DNA of interest’
2.Design primers with restriction site sequences included at 5’ ends
3.Amplify ‘DNA of interest’ using PCR
4.Digest both ‘DNA of interest’ and plasmid with chosen restriction enzymes
5.Combine digested ‘DNA of interest’ and plasmid, ligate using DNA ligase
DNA Ligase
Capable of joining together compatible ends of DNA molecules by joining the sugar-phosphate backbone
Target DNA can be joined to vector DNA following overlap of sticky ends or by two sections joining at blunt ends
How do we get recombinant DNA into the cell?
Transformation methods - Make cells competent
Heat Shock - Chemically competent cells
Electroporation - Electrocompetent cells
