Unit 2 Flashcards
What is a restriction enzyme? What does it do?
type of endonuclease enzyme - cleaves phosphodiester bonds between nucleotides and DNA
basis for DNA cloning and recombinant DNA technology
Which type of restriction enzyme cuts remote from the recognition sequence?
Type 1
Where does a Type 2 restriction enzyme cut?
cuts within recognition sequence
Modified DNA is cut by which type of restriction enzyme?
Type 4
What does a type 3 restriction enzyme cut?
cuts near the recognition sequence
Why is a 5’ or 3’ overhang desirable?
can rejoin sequences that have been digested by the same enzyme
How does DNA ligase work?
What does it require?
rejoins compatible sticky ends by creating new phosphodiester bonds- requires ATP!
What is a plasmid?
Circular piece of DNA that are independent from the bacterial chromosome.
What are the advantages of cloning in Plasmids?
Generates more DNA
Can amplify larger DNA fragments
Generally stable
Enables expression of gene to produce recombinant proteins
How do you clone a fragment of DNA into a plasmid?
When a DNA fragment has the same restriction sites on either end, restriction enzyme cuts the fragment and digests the plasmid leaving the sticky ends.
2. These sticky ends during ligation create a new circular DNA plasmid containing fragment.
When is directional cloning used?
when two different restriction enzymes with different recognition sites give different sticky ends at each end of the fragment and plasmid.
How does Directional Cloning work?
- Restriction enzymes digest both of the DNA to create sticky ends.
- DNA ligase is added and then mixed with competent bacteria (E.Coli) - will create transformed and untransformed cells.
What are the names of each step in the PCR process? (4)
- Denaturation
- Annealing
- Elongation
- Final Elongation
What occurs in the Denaturation steps of the PCR reaction?
heat to 94 degrees to break the hydrogen bonds and seperate the strands
Why in the PCR reaction after denaturation is the substance cooled?
allows the primers to anneal to the sequence
What are the primers referred to as?
oligonucleotide primers
What occurs in the elongation stage of the PCR?
Temperature rises (optimum for Taq polymerase), Taq adds nucleotides to the 3’ hydroxyl of the primers
What is distinctive about the first elongation stage?
[hint- what does the Taq do?]
Taq extends the strand past the amplicon on the first round
What are the two methods used for DNA Sequencing?
Sanger Sequencing
Next generation sequencing
Why sequence DNA?
can determine if a cloned fragment or PCR product is correct
detects mutations
establishes relatedness of organisms.
What are the steps to Next generation sequencing?
- Library Preparation- DNA is fragmented down to nucleotides & adapters added.
- Cluster amplification- adaptor sequences can then anneal to oligonucleotides which are amplified and copied.
- Sequencing- utilises a fluorescent nucleotide
- Alignment & Data analysis
How is Next Gen sequencing used in clinical practice?
If a child is born with a particular disease, doctors can take a DNA sample, sequence the genome of the child and parents, analyse the variance and find the cause of the disease.
What are the limitations of Next Gen sequencing?
Generates huge amount of data and requires special software to interpret it correctly.
How does Sanger sequencing work?
Cycles through multiple rounds of PCR reaction to create multiple DNA fragments- each dideoxynucleotide has a different fluorescent tag which we can use in gel electrophoresis to determine the length of the fragments.
In a sequencing trace the height of the peaks represent 1.________
Colour of the peaks determines 2.__________
- fluorescence intensity that is detected
2. the dye detected (and therefore the specific deoxynucleotide base).
What is Transgenics?
an organism that contains genetic material into which DNA from an unrelated organism has been artificially introduced.
an animal with altered DNA
Give Examples of Transgenic Experiments?
Overexpression or ectopic expression
Promoter Analysis
Gene Downregulation
Gene therapy
What is the role of a transgenic expression construct?
helps keep transgenic expression constant
What are the advantages to Transgenics?
fast assessment of gene function
creates tools for specific gene regulation studies
What are disadvantages of Transgenics?
Site of integration can modify gene expression
Large number of animals are used to produce a Transgenic one
Integration into the genome is random.
How does Gene Targeting differ from Transgenics?
Transgenics is animals with altered DNA, random process, uses more animals but produces informative subjects first.
Gene Targeting is where a DNA Segment is introduced into the cell, specific but usually a slower process.
What is Cre loxP Recombination? What is it used for?
site specific recombination used to carry out deletions, insertions, translocations at specific sites.
What are the requirements for Cre loxP recombination?
need isogenic DNA (identical genes)
need to know the gene being targeted.
What are the steps in Cre loxP Recombination?
- region of DNA to be targeted is flanked by loxP sites
- Cre recombinase is introduced to catalyse the following reaction.
- Cre protein is produced in the tissue of interest, Cre mediated recombination between loxP sites deletes intervening DNA.
- The leftover gene function is disrupted in the tissue.
