CHAPTER 2: DNA MANIPULATION Flashcards
1
Q
DNA polymerase
A
uses DNA polymerase to accurately copy a DNA template
2
Q
endonucleases - restriction enzymes
A
- restriction enzymes like endonucleases cut DNA at specific recognition sequences known as restriction sites, splitting DNA into smaller fragments
- restriction sites is a particular order of nucleotides
- endonucleases make one incision on each of the 2 complementary strands of DNA
3
Q
sticky ends
A
- endonucleases cut one strand at one point but cut the second strand at a point that is not directly opposite → causes an overhang to form
- DNA ligase enzyme connects the single-stranded DNA together via the sugar-phosphate back bones
4
Q
which pairs faster, sticky or blunt
A
- pieces of DNA with sticky ends pair faster than pieces of DNA with blunt ends
- sticky ends allow for complementary base pairing so the pieces are held together by weak hydrogen bonds and can be acted on by the DNA ligase enzyme.
5
Q
blunt ends
A
- endonuclease cuts the 2 strands of DNA molecule at points DIRECTLY OPPOSITE each other to produce cut end
- DNA fragments are joined directly together through the use of DNA ligase
6
Q
DNA ligase
A
- an enzyme known as ligases catalyses the joining of pieces of double-stranded DNA at their sugar-phosphate bones
7
Q
what are plasmids
A
- circular, double stranded DNA that can reproduce independently
- can be taken up by bacterial cells
- many are used as vectors to transport foreign DNA into bacterial cells to transform them
- they have multiple recognition sites (restriction sites) for endonucleases
- can have new genes inserted
- have an antibiotic resistant marker (selective marker)
- have a promoter or origin point for self replication
8
Q
making recombinant plasmids
A
- cut plasmid DNA with endonuclease
- cut foreign DNA with the same endonuclease
- mix plasmids and foreign DNA
- add DNA ligase which joins the sticky ends together
- same recognition site allows same restriction enzyme to cut both the plasmid and the gene of interest
- produces matching sticky ends on the plasmids and the gene of interest
- plasmids will then join with the DNA of the gene of interest to form recombinant plasmids as they have complementary sticky ends.
- the two fragments can then be successfully joined by DNA ligase.
9
Q
recombinant plasmid
A
a plasmid that has taken in new DNA is called a recombinant plasmid
10
Q
bacterial transformation
A
- when bacteria cells take up plasmids
- recombinant plasmids need to be taken up by bacteria
- techniques that temporarily interfere with the plasma membrane allow this (causes holes in the pm so plasmid can enter)
- electroporation
- heat shock
11
Q
how is transformed bacteria identified
A
- bacteria can take up recombinant plasmid with AmpR gene
- results in bacteria resistant to ampicillin antibiotic
- bacteria can take up recombinant plasmid with green fluorescent protein (gfp gene_
- when uv light is used, bacteria is fluorescent
- but needs arabinose , sugar to repress the repressor of the gfp gene
12
Q
ethical issues of gene cloning
A
- changing a species’ DNA may result in unforeseen consequences
- concern that the pharmaceutical product may contain bacteria that will cause disease
- not natural and therefore may be against religious or moral views
- respect → embryos cannot give informed consent
- respect → some believe that modifying embryos does not honour the sanctity of life
- justice → gene editing technology is expensive and may only be available to wealthy people
- non-maleficence → treated individuals or embryos may experience unforeseen side effects
13
Q
purpose of LB broth
A
- provides nutrients to support bacterial growth
- both transformed and untransformed bacteria can grow
14
Q
purpose of ampicillin
A
- to identify which bacteria has been transformed
- bacterial growth → has the +pREC because it has AmpR gene, resistant to ampicillin antibiotic
- no bacterial growth → is -pREC no AmpR gene
15
Q
purpose of arabinose
A
- to also identify which bacteria has been transformed
- arabinose represses the repressor of the GFP gene (binds to protein AraC)
- allow the GFP gene to be expressed
- so under UV light, bacteria will appear green
16
Q
potential errors
- transforming bacteria experiment
A
- bacteria was left in the heat bath for too long, killing the bacteria before plating it
- the wrong test tube was put on the plate
- no recomb test tube was added to the wrong plate (plates mixed up) → human error
17
Q
benefits of recombinant insulin
A
- high levels of purity
- reiability of supply
- reduced chance of side effects such as allergies (compared to pig or cow derived insulin)
- consistency of quality between batches
18
Q
gel electrophoresis
A
- a method used to separate DNA fragments based on size (length, measured in bp)
- the phosphate group of nucleotides is negatively charged
- negatively charged DNA moved towards the positive terminal (b/c it is negatively charged)
- DNA is loaded into a gel that acts like a sieve to allow SMALLER DNA fragments through more quickly than larger ones
- the result of gel electrophoresis is a series of PARALLEL BANDS of DNA fragments at differing distances down the gel
- each band can contain millions of DNA molecules of the same size
19
Q
applications of gel electrophoresis
A
- people have DIFFERENT DNA SEQUENCES, so when endonucleases are used, the DNA will be cut at DIFFERENT LOCATIONS and DNA fragments of different lengths will result
- gel electrophoresis can identify people in:
- forensic investigations
- mass disasters
- paternity testing
- identifying animals
20
Q
mitochondrial DNA
A
- not all DNA is used in gel electrophoresis
- nuclear or mitochondrial DNA can be used
- compared to nuclear DNA, mtDNA
- is inherited via the MATERNAL LINE
- does not recombine during reproduction → LESS VARIABLE
- is present in LARGER AMOUNTS
21
Q
dna profiling - short tandem repeats
A
- chromosomal sites (non coding region) where many copies of a short DNA sequence are joined end-to-end; the number of repeats is variable between unrelated people
- STRs are 2-5 bp in length and repeated over and over
- the number of REPEATS VARIES between people and each variation is a distinct allele
- we have 2 copies of each STR - one from mother and one from father
22
Q
types of DNA used for DNA profiles
A
- to develop DNA fingerprints or DNA profiles, regions with high variation between individuals are used.
- Short Tandem Repeats (STRs) from nuclear DNA
- STRs can identify individuals
- Hypervariable regions (HVRs) in mtDNA
- mtDNA can identify relationships/when fewer cells are available
- Short Tandem Repeats (STRs) from nuclear DNA
23
Q
bacteriophages
A
- there are viruses called bacteriophages
- they inject viral DNA into the bacteria
- they hijack the bacteria’s cellular machinery to reproduce to make more bacteriophages
- this causes the bacteriophage to burst out and cycle repeats to infect more bacteria
24
Q
CRISPR regions
A
- CRISPR is an immune response found naturally in bacteria and is modified in the lab to edit genes
- CRISPR provides a form of NATURAL IMMUNITY for the bacteria against viral attacks
- DNA with short repeated sequences (repeats) and spacers
- CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS
- each repeat is separated by a ‘spacer’ DNA
- the spacer DNA is a segment of DNA from bacteriophages the cell has encountered previously
- these clusters are regularly interspaced with the repeat DNA
25
spacers
- are made up of remnants of DNA from bacteriophages of foreign DNA
- when bacteria encounters a new virus, they store some of its DNA in a new spacer sequence
26
how does CRISPR-Cas9 work
- a bacteriophage attaches to the outside of bacterial cell and injects its VIRAL DNA into the cell (reinfection)
- previously, a segment of the viral DNA has been stored as a spacer in the CRISPR region
- the CRISPR sequence is TRANSCRIBED resulting in pre-CRISPR RNA (crRNA)
- tracer RNA (trcrRNA) has a complementary sequence to the repeat DNA (NOT the spacers)
- role: helps hold the gRNA in place in the Cas9 enzyme
- RNAase cuts spacer, portion of the repeat and associated tracr-RNA to become g-RNA
- the specific spacer of the crRNA binds to the trcrRNA to form guide RNA (gRNA)
- gRNA then binds with the Cas9 enzyme → forms a **Cas9-gRNA complex**
- Cas9-gRNA complex scans bacteriophage (target DNA) to look for complementary bases
- once it is found, the DNA is unzipped and Cas9 **cuts/cleaves** the DNA creating **blunt DNA fragments.**
- DOUBLE BLUNT END CUT
- the viral DNA cannot reproduce as the DNA has been **disrupted**
27
uses of CRISPR Cas9
- scientists can program CRISPR-Cas9 system to TARGET ANY SEQUENCE using the gRNA
- can introduce a sgRNA (single guide RNA) that is complementary to the gene of interest
- **knock out** genes one at a time, in order to identify their function
- to **introduce** specific mutations in a DNA sequence
- to **edit** a faulty allele of a gene in a person with a severe inherited disease
- to **snip out** the faulty segment of a gene and replace it with a working copy
- to **activate** or to **repress** a gene
- to **add** a new gene to the genome
## Footnote
it is an easy and cost-effective way of editing an organism's genome
28
what is PCR used for
- used to **amplify** (make more copies) of a segment of DNA
- process depends on the enzyme DNA polymerase → specifically Taq polymerase
- doubles the amount of DNA with EACH CYCLE
29
steps in PCR
- denature
- the DNA sample is heated **94 degrees to 98 degrees for one minute**
- this breaks the hydrogen bonds between the double strands
- separates DNA into two single, separate strands
- anneal
- PRIMERS are added
- short segments of SINGLE STRANDED DNA
- primers bind to the target DNA (at the complementary sequences), initiating DNA synthesis **50 degrees to 65 degrees for two minutes**
- extension
- taq polymerase starts at the primers and extends them using free nucleotides
- forms TWO COMPLETE DOUBLE STRANDS
- occurs at around **72 degrees for one minute** → optimal temperature of taq polymerase
- the process is then repeated
30
ethical requirements of human experimentation
- Respect for persons: Informed consent
- E.g. people must know what they are agreeing to (harms, likelihood of success, etc.)
- Integrity: Being honest and trustworthy
- E.g. Being open about the likelihood of benefit or harm
- Justice: Selection of human subjects and non-discrimination
- E.g. vulnerable people not targeted
- Beneficence: Maximising benefit and minimizing harm
- E.g. the welfare of the person is most important (the research comes second)
- Non-maleficence – do no harm
- Eg. Stopping a treatment if it is causing harm
31
CRISPR Cas9 ethical considerations
- long-term impacts of the technology and informed consent risks
- possibility of the edited genes being inherited and affecting future generations
- the use in individuals who may have not provided consent
- misusing technology such as around the use for designer babies
- confidentiality considerations of genetic data
- personal beliefs and opinions around the editing of human DNA (such as religious or cultural objections).
32
how to run a gel electrophoresis
1. DNA sample is combined with DNA loading dye.
2. the mixture is placed in a well at one end of the agarose gel.
3. the agarose gel is immersed in a buffer solution (salt solution).
4. it is then exposed to an electric field with the positive (+) pole at the far end and the negative (−) pole (cathode) at the well
5. smaller fragments move through the agarose gel faster compared to larger fragments.
6. these fragments appear as bands on a gel which can be interpreted in various ways. This usually needs to be observed under UV light.
33
how can CRISPR Cas9 be programmed to cut any DNA sequence
- scientists create a complex, consisting of a gRNA sequence and the Cas9 protein
- gRNA sequence is complementary to the target DNA sequence
- Using the gRNA, Cas9 identifies the complementary DNA sequence and unwinds the DNA and cuts it
- scientist can insert a new piece of DNA, remove, replace or add or delete single nucleotides at the site of the cut
- the cell detects and repairs the broken strands of DNA
34
purpose of the PAM sequence
* a highly specific region
* **a very short nucleotide sequence adjacent to the target spacer** (PAM sequence)
* using gRNA, cas9 enzyme searches viral DNA for a PAM
* upon finding it, CAS9 complex compares the sequence of bases in the crRNA to a sequence upstream of the PAM
* If they are complementary, Cas9 cuts the DNA, preventing infection.
* also where Cas9 binds to on the target DNA sequence
* plays an essential role in **distinguishing self bacterial DNA from non-self viral DNA**
35
purpose of Cas9 enzyme
- Cas9 will only cut out the target sequence if it recognises a very short nucleotide sequence adjacent to the target spacer called a protospacer adjacent motif (PAM) sequence.
- Cas9 is an endonuclease associated with CRISPR and acts likes a pair of molecular scissors capable of precisely cutting both strands of DNA.
36
GMO definition
genetically modifies organisms are organisms that have had their DNA directly manipulated
37
transgenic definition
- transgenic organisms are a subset of GMOs that have DNA from different species
- eg. transforming bacteria with recombinant plasmid
38
how GMO can increase crop productivity
- roundup ready canola
- canola plants are used to make canola oil
- roundup is a herbicide
- roundup ready canola has a bacterial gene inserted
- this allows the canola plant to make an enzyme to break down the herbicide roundup
- GM canola crops can be sprayed with the herbicide and only the weeds die, leaves the GM canola crops unaffected
39
how GMO can provide pest resistance
- Bt cotton has a bacterial gene that allows it to produce chemicals that will kill insects
- the crops are not eaten by insects → resulting in more cotton production
- crops do not need to be sprayed with insecticide
40
compare genetic modification to other methods
- faster (traditional techniques like artificial selection took time to breed the organisms with the favourable traits)
- more precise (edit specifically the DNA for the trait desired)
41
what is more useful for gel electrophoresis- sticky or blunt ends
* blunt ends
* sticky ends may allow for the hydrogen bonds between the complementary base pairs to reform
* causes fewer bands on the gel - less reliable results
42
making synthetic insulin
- human insulin is composed of **2 polypeptide chains**
- an A chain and a B chain. It is therefore a quaternary structure.
- the A chain and the B chain are synthesised separately on **separate plasmids in separate bacteria**.
- the plasmid used has antibiotic resistance selectable markers to allow for recombinant plasmid selection.
- the insulin chain that is inserted into the plasmid has been modified to **remove all introns** as prokaryotes don’t have Introns.
- the insulin genes are inserted **next to a gene for β-galactosidase protein**, which allows for **detection of successful gene insertion.**
- The beta gal gene has 2 functions
- its an additional selectable marker
- the beta gal gene is an inducible operon and therefore can regulate A chain production
- **expression of each gene** from the two bacteria allows a functional fusion protein to be produced.
- once the genes are expressed and the fusion proteins are produced by each bacteria, these **fusion proteins are then purified**
- the insulin polypeptides are removed and then **combined together to produce functional insulin.**
43
difference between Cas9 and a typical endonuclease
* Cas9 requires a **PAM sequence adjacent** to the target site in order to cleave
* typical restriction enzymes cut at a set **recognition site** sequence, whereas Cas9 cuts at any target DNA sequence that is designated by single guide RNA (target DNA sequence is complementary to the sgRNA)
44
difference between gRNA and sgRNA
* gRNA (guide RNA) is comprised of 2 RNA molecules: crRNA and tracrRNA
* scientists manufacture a synthetic sgRNA (single guide RNA) that is comprised of 1 RNA molecule
sgRNA serves as both the tracrRNA and crRNA (containing the spacer)
45
the role of primers and why we need to have two DIFFERENT primers
* A primer attaches to complementary DNA nucleotides.
* The addition of the primer and its joining to complementary nucleotides then allows the DNA polymerase to begin copying.
* Two primers are needed as the nucleotide sequence is different at the start and finish of the section of DNA that must be copied.
46
function of ORI on a plasmid
the site at which the plasmid replicates
47
how can gel electrophoresis be used to determine if a plasmid is recombinant?
* Adding foreign DNA to make a plasmid recombinant would increase its size.
* when run through the gel electrophoresis you can then compare the banding pattern of the plasmid sample to the control sample and DNA ladder of known sizes
* If you see additional bands in the plasmid sample that are **not present in the control**, these bands correspond to the **insert DNA**, indicating that the plasmid is recombinant.
