Term 2 Lecture 3: DNA Cloning, The Basics Flashcards
Cloning in the context of recombinant DNA
X we’re not talking about cellular cloning like Dolly the sheep
✓ we’re focusing on recombinant DNA isolation of pieces of DNA and cloning them to produce multiple copies in enough quantity to perform studies - mostly bacterial plasmid work.
Recombinant DNA technology definition
The joining together of DNA molecules from 2 different species. Recombined DNA is inserted into a host to produce new genetic combinations of value to science, medicine, agriculture and industry
Why is recombinant DNA useful?
For understanding the function of genes and the proteins they encode:
- isolating a gene so its sequence can be determined
-isolating a gene, mutating it and looking for an altered phenotype (reverse genetics)
- or by introducing a wildtype copy of a gene into a mutant to see if it rescues a mutant thereby confirming the nature of the mutation
- to follow the activity of genes and proteins by linking them to ‘reporter genes’
- in biotech recombinant DNA can be designed to produce valuable substances
Biotech applications
A colour code exists:
Red- applied to medical processes
Production of recombinant proteins e.g. insulin
White- more efficient industrial processes
E.g. enzymes for scouring cotton - microbial fermentation that produces enzymes that replace chemicals
Green- applied to agriculture
Crop improvement e.g. nutrient supplementation increasing vitamin A content in golden rice or increasing salt tolerance of plants by adding a gene to move salt to a vacuole to allow growth.
Blue: marine and aquatic applications
E.g. ziconotide a painkiller >60x stronger than morphine derived from cone snail venom
Yellow: novel products from insects
Recombinant silk producing via a microbial system to produce silk without insects or use of spider venom for pesticides
What is needed for DNA cloning and basic process
1) enzymes for manipulating DNA
2) a cloning vector/cassette
3) biological host cells
4) a gene you’d like to clone
Process
Introduce dsDNA recombinant plasmid containing your chosen genes to a bacterial cell and culture it to produce hundreds of millions of the bacteria then harvest recombinant plasmids from lysed bacterial cells to insert into hosts.
The majority of cloning experiments are carried out in bacteria most commonly E. Coli even if the final aim is to express the genes in a plant or animal (this is key in designing cloning experiments)
DNA cloning step 1: find enzymes for manipulating DNA
Enzymes used for manipulating DNA:
1) cutting (restricting)
restriction enzymes
2) joining (ligating)
ligase enzymes
3) synthesising (amplifying)
DNA polymerases, reverse transcriptase
More on Restriction enzymes (RE)
RE are bacterial enzymes that provide protection from viruses (bacteriophages)
RE have individual recognition sequences which they “find” rapidly in complex DNA mix
Recognition sequences are usually 4-8 nucleotides and palindromic (read the same forwards and backwards)
e.g. 5’ CCGCGG 3’
Some REs cut in a staggered way producing sticky ends with one ssDNa strand overhanging whilst others produce blunt ends
Both sticky and blunt ends can be stuck back together with DNA ligases
Following RE digestion it’s very common to run DNA molecules on agarose gel to check the DNA has been cut (gel electrophoresis)
DNA cloning step 2: cloning vector/cassette
- usually plasmids exist in plants, animals and prokaryotes but only bacterial plasmids are used in cloning
- plasmids are easy to isolate, circular and exist in many conformations - to find their size they must be linearised (cut once with a RE such as EcoR1)
- plasmids are easy to reintroduce (once transformed) back into bacterial cells
Bacterial plasmids as cloning vectors:
A) cloning vector must contain OriC region: origin of replication that the host cell recognises to replicate it along with the DNA it carries.
B) they should carry selectable markers - traits that enable cells containing the vector to be selected/identified
C) a cloning vector needs a single cleavage site for each of the one or more restriction enzymes used..
steps of cloning:
1) plasmid and foreign DNA cut by same enzyme
2) when mixed sticky ends anneal joining foreign DNA and plasmid
3) nicks in sugar-phosphate bonds are sealed with DNA ligases to form a circle
The sticky ends of the vector can ligase back together so many vectors are designed so that differentiating between recombinant clones and ‘empty’ plasmids is easy
How can we differentiate between recombinant and empty plasmids?
By interrupting a gene with the foreign gene you’re introducing e.g. cloning RE site in the middle of lac2 gene a gene that codes for a peptide required to produce functional beta galactosidase enzyme - this enzyme breaks down x-gal to form a blue dye
So if you clone DNA into the reading frame of the lac2 gene it is disrupted and no enzyme is made therefore these colonies when plated with x-gal produce no blue dye. So white colonies on the gel are recombinant and the blue ones are ‘empty’ the recombinant colonies can then be transferred for incubation and the empty colones discarded.
Each colony on a Petri dish grows from one bacterium
How do we make bacteria competent?
Competent bacteria are able to take up recombinant plasmids.
To .ake them competent they are treated with a salt solution usually CaCl or RbCl which makes the bacterial cell membranes more permeable (and fragile).
The bacteria are kept on ice to prevent them from bursting
just after adding plasmids they are heat shocked at 42°C for 30secs to open pores
After heat shocking growth media is added to allow recovery, the cell walls heal and recombinant plasmids in cells are replicated
They are then plated with x-gal and antibiotic (to grow only resistant cells)
White colonies are recombinant and blue are empty
