Lecture 3 Flashcards
(38 cards)
What are the two main methods for DNA cloning?
Cell based
Polymerase chain reaction
What is used to carry the DNA fragment into the host cell?
Vectors
PCR
Polymerase chain reaction - selectively copies a desired sequence present in a complex mixture of other DNA molecules
Describe cycle 1 of PCR
95 breaks down hydrogen bonds between complementary strands. Strands separate
60 Allows short oligonucleotide primers to anneal, specific to the 3’ end
72 Primers guide Taq polymerase (thermostable) which synthesises complementary strands from free phosphorylated nucleotides
Two molecules of DNA containing the target sequence
Describe cycle 2 of PCR
Repeat stages.
4 partially double stranded molecules each containing the target sequence
Describe cycle 3 of PCR
Repeat stages
2 double stranded molecules containing only the target sequence and 6 longer length molecules containing the target sequence and adjacent nucleotides.
In PCR how does the amount of target sequence grow with each cycle?
Exponentially
What is DNA cloning used for?
Produce DNA libraries
Sequence DNA
Simple DNA molecules can be amplified allowing it to be…
Studied/separated
Manipulated- mutagenised or engineered
Expressed- generation of protein
What are the 5 steps in the plasmid cloning strategy
Enzyme restriction: digests a DNA sample
Enzyme restriction: Digest of DNA plasmid vector
Ligation of DNA sample products and plasmid vector
Transformation with the ligation products
Growth of agar plates with selection for antibiotic resistance
Restriction enzymes
Enzymes that cut DNA in specific places
Inactivate foreign DNA
Break palindrome sequences (those exhibiting two fold symmetry)
Important in DNA research (hybridisation)
What are the types of different ends produced by restriction enzymes?
5’ overhangs: the enzyme cuts asymmetrically within the recognition site such that a short single stranded segment extends from the 5’ end
3’ overhang: the enzyme cuts asymmetrically within the recognition site such that a short single stranded segment extends from the 3’ end
Blunt: enzymes cut at ipposite sites in the two strands of DNA and generate blunt ends without overhangs.
Briefly describe the 5 steps of the cloning process
Gene of interest cut out using a restriction enzyme
Host plasmid is cut with the same restriction enzyme
New plasmid inserted into bacterium (transform)
Grow host cells under restrictive conditions, grow on plates containing an antibiotic
Directional cloning
Inserting foreign DNA in a particular orientation done by making two cleavages with two different restriction enzymes so the foreign DNA can only be inserted in one direction.
The vector and foreign DNA are cut with the same 2 different restriction enzymes, this produces cohesive/sticky ends. The DNA can be ligated in only one direction and there is a low background of non-recombinant plasmids.
How can you increase efficiency in cloning?
When one restriction enzyme is used to cut the vector and insert = low efficiency.
DNA can be inserted in two directions and tandem copies of inserts may be found.
What can be used to avoid large amounts of non-recombinant plasmids?
Alkaline phosphate- it removes the 5’ phosphate groups from the cut vector to prevent self-ligation.
Most active at alkaline pH.
What are the two main uses of alkaline phosphatase in DNA manipuation?
Removing 5’ phosphates from plasmid and bacteriophage vectors that have been cut with a restriction enzyme. Prevents self-ligation
Removing 5’ phosphates from fragments of DNA prior to labelling with radioactive phosphate. Polynucleotide kinase is much more effective in phosphorylating DNA if the 5’ phosphate has previously been removed.
Directional cloning, step 1
Re-digestion of DNA sample
DNA sample cut by ECOR 1 which produces many fragments
Directional cloning, step 2
Re-digestion of plasmid DNA
The plasmid is circular and can survive outside of the E.coli cell.
The plasmid is double stranded circular DNA and contains an ampicillin resistant gene (AMPR)
This gene can be seleted.
Multiple cloning site allows enzymes to cut it. The MCS is in the middle of Lac Z
Lac Z produces B-galactosidase which breaks down X-gal to produce a blue/purple colour. If the gene is inserted then the Lac Z gene is disrupted and the enzyme is disrupted and the enzyme is not made and X gal is not broken down so white colour is seen.
Directional cloning, step 3
DNA ligase joins the DNA to the plasmid by forming phosphodiester bonds, hydrogen bonds form between nucleotides.
Directional cloning, step 4
Transformation of ligation products. Aim is to make pores in the membrane of the E.coli cell so the DNA can enter.
The recombinant plasmid must enter the E.coli cell.
There are two main methods to do this:
chemical method using CaCl2 and heat shock to promote DNA entry into cells
Electroporation based in a short pulse of electric charge to facilitate DNA uptake
What is the process of transferring exogenous DNA into cells
Transformation
Chemical transformation with calcium chloride
Chill on ice => mix with liquid nitrogen to store at 80 degrees
Water bath for 90 seconds
Spread on a plate of agar which contains ampicillin and X gal and the purple cells that frow are those that have not taken up (lac z still intact). The non-ampicillin resistance cells die.
Transformation by electroporation
Most efficient/effective method
Put plate in an incubator at 37 degrees
Leave overnight
Observe results
