CHAPTER 2: DNA MANIPULATION Flashcards
1
Q
DNA polymerase
in DNA manipulation
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
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
8
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
9
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
10
Q
dna profiling - short tandem repeats
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
11
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
12
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
13
Q
making recombinant plasmids
A
- cut plasmid DNA and 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.
14
Q
recombinant plasmid
A
a plasmid that has taken in new/ foreign DNA
15
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
16
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
17
Q
purpose of LB broth
A
- provides nutrients to support bacterial growth
- both transformed and untransformed bacteria can grow
18
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
19
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
20
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
21
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
22
Q
how to run a gel electrophoresis
A
- DNA sample is combined with DNA loading dye.
- the mixture is placed in a well at one end of the agarose gel.
- the agarose gel is immersed in a buffer solution (salt solution).
- it is then exposed to an electric field with the positive(+)pole at the far end and the negative(−) pole (cathode) at the well
- smaller fragments move through the agarose gel faster compared to larger fragments.
- these fragments appear as bands on a gel which can be interpreted in various ways. This usually needs to be observed under UV light.
23
Q
what is more useful for gel electrophoresis- sticky or blunt ends
A
- sticky ends facilitate ligation by DNA ligase by forming hydrogen bonds between complementary bases of the other strand.
24
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
25
CRISPR regions
* 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
26
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
27
how does CRISPR-Cas9 work
1. 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
2. the CRISPR sequence is TRANSCRIBED resulting in pre-CRISPR RNA (crRNA)
3. 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
4. RNAase cuts spacer, portion of the repeat and associated tracr-RNA to become g-RNA
5. the specific spacer of the crRNA binds to the trcrRNA to form guide RNA (gRNA)
6. gRNA then binds with the Cas9 enzyme → forms a **Cas9-gRNA complex**
7. Cas9-gRNA complex scans bacteriophage (target DNA) to look for complementary bases - PAM SEQUENCE
8. once it is found, the DNA is unzipped and Cas9 **cuts/cleaves** the DNA creating **blunt DNA fragments**.
9. DOUBLE BLUNT END CUT
10. the viral DNA cannot reproduce as the DNA has been **disrupted**
28
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
29
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
30
denature
| steps of PCR
* 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
31
anneal
| steps of PCR
* 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**
32
extension
| steps of PCR
* 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
33
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
34
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).
35
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
36
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**
37
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.
38
GMO definition
genetically modifies organisms are organisms that have had their DNA directly manipulated
39
transgenic definition
* transgenic organisms are a subset of GMOs that have DNA from different species
* eg. transforming bacteria with recombinant plasmid
40
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 that 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
41
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
