ch 4 sac revision (4A-D, 4F) Flashcards
1
Q
what are endonucleases
A
refer to a broad range of enzymes responsible for cutting strands of DNA.
2
Q
what is a restriction endonuclease
A
targets a specific recognition site
3
Q
how do enzymes “cut” the dna?
A
the enzymes cleave the phosphodiester bond of the sugar phosphate backbone that holds the dna nucleotides together
this process is sometimes called “restriction endonuclease digestion”
4
Q
where are restriction endonucleases sourced from
A
from bacteria, where they are naturally produced as a defense against invading viral DNA that could harm the bacteria
5
Q
how are restriction endonucleases named
A
in the bacteria that they’re discovered in
6
Q
how long is a restriction site of an endonuclease
A
usually 4-6 nucleotides in length -> specific to each enzyme
7
Q
what does it mean when the recognition sites are palindromes
A
the 5’ to 3’ sequence of the template strand is the same as the 5’ to 3’ sequence of the non - template strand
8
Q
sticky ends
A
do not cut in the middle of the recognition site, resulting in a staggered cut resulting in overhanging, unpaired nucleotides
9
Q
blunt ends
A
cut DNA in the middle of the recognition site, which results in a straight cut and no overhanging nucleotides
10
Q
why are sticky ends beneficial
A
the endonucleases have the advantage of ensuring an inserted gene is orientated correctly when manipulating DNA
11
Q
what are ligases
A
enzymes that join 2 or fragments of DNA or RNA like molecular glue
12
Q
how does ligase join 2 fragments of DNA or RNA together
A
ligase will catalyse the formation of phosphodiester bonds b/n the 2 fragments to merge them
13
Q
2 types of ligase?
A
DNA ligase - joins 2 DNA fragments
RNA ligase - joins two RNA fragments
14
Q
what are the ligases enzymes function the opposite of
A
endonucleases but they lack the restriction of endonuclease so they can join both blunt and sticky ends
15
Q
why are ligases able to join both blunt or sticky ends
A
This is because the substrates for this enzyme are the sugar and phosphate
groups of the DNA or RNA, rather than specific nitrogenous bases which is the case for restriction endonucleases.
16
Q
what are polymerases
A
add nucleotides to DNA or RNA, which can lead to copying entire genes. AKA synthesise polymer chains from monomer building blocks
17
Q
2 types of polymerases used for gene manipulation
A
RNA and DNA polymerase
the monomer would be a DNA or RNA nucleotide
the polymer would be a DNA or RNA strand
18
Q
what is rna polymerase used for
A
transcription of genes
19
Q
what is DNA polymerase used for
A
amplification of DNA
20
Q
give an example of DNA polymerase
A
For example, in forensic medicine, when
scientists are testing a sample they often have a very small amount of DNA available. DNA
polymerase can be used to synthesise more strands of DNA, thereby amplifying the DNA.
21
Q
what do polymerases req
A
a primer to attach at the start of a template strand of DNA.
22
Q
what are primers
A
short single stranded chains of nucleotides that are complementary to the template strand
23
Q
what happens when the primer is attached to the template strand
A
once attached to the primer, the polymerase enzyme can read and synthesise a
complementary strand to the template strand in a 5’ to 3’ direction
24
Q
what is crispr
A
is a naturally occurring sequence of DNA found in bacteria that plays an important role in their defence against viral attacks
25
how does the bacterium recognise a virus
When a bacterium encounters a virus, it takes a ‘mugshot’ of it by storing some of the
viral genetic material within the bacterium’s own genome. Next time the virus invades,
the bacterium transcribes the ‘mugshot’ DNA and attaches it to an endonuclease called
Cas9. T
26
how does cas 9 only destroy the invading virus
. The transcribed mugshot is complementary to the viral DNA, so it ensures that
the Cas9 only destroys the invading virus rather than any bacterial nucleic acids. In this
way, we can say that CRISPR-Cas9 functions as a primitive adaptive immune system in
bacteria, defending them from viral invasion.
27
how is it recognisable in the bacterial genome
The system is relatively easy to recognise in the
bacterial genome: it is a section of DNA with short, repeated sequences of nucleotides
that have the same forward and reverse read. In other words, Clustered Regularly
Interspaced Short Palindromic Repeats (CRISPR)
28
The clustered repeats all what...
The clustered repeats
are interrupted by spacer DNA, which is the viral ‘mugshot’ of DNA cut from invading
bacteriophages that allows for recognition during subsequent invasions. CRISPR
sequences are always downstream of the gene for Cas9.
29
3 steps to fighting the virus with crispr
Exposure, expression, extermination
30
describe exposure
the bacteriophage injects its DNA into a bacterium, which identifies
the viral DNA as a foreign substance. Cas1 and Cas2 are both CRISPR-associated
enzymes like Cas9, but they serve a different purpose. These enzymes cut out a short
section of the viral DNA (typically ~30 nucleotides long), known as a protospacer.
This protospacer can then be introduced into the bacterium’s CRISPR gene and
become a spacer (Figure 3).
31
describe expression
Expression – the CRISPR spacers are transcribed along with half a palindrome
from the repeat either side of it, and converted into an RNA molecule known as
guide RNA (gRNA). gRNA binds to Cas9 to create a CRISPR-Cas9 complex which
is directed to any viral DNA inside the cell that is complementary to the gRNA
(Figure 5). gRNA forms a hairpin loop-like structure from the transcribed
palindromic repeats either side of the spacer.
32
describe extermination
- The CRISPR-Cas9 complex then scans the cell for invading
bacteriophage DNA that is complementary to the ‘mugshot’ on the gRNA. When it
does, Cas9 cleaves the phosphate-sugar backbone to inactivate the virus. Cas9 contains
two active sites to cut both strands of DNA and create blunt ends (Figure 7)
33
what happens when the viral DNA is cut
enzymes within the bacterium will naturally act to repair it.
However, the repair mechanisms in a cell are prone to errors that can result in nucleotide
additions, deletions, or insertions in the middle of the viral gene.
34
why are these repair mechanisms advantageous
This is advantageous in
the case of bacteriophage infiltration because these mutations tend to render viral genes
non-functional. If a mutation does not occur after the cut, the gRNA will find the gene
again and repeat the whole process until the DNA repair mechanisms induce a mutation,
inactivating the virus
35
what can genetic modifications do, why is this good/bad/both
Genetic modifications can amend deleterious mutations or introduce biologically
advantageous alleles to an individual’s genome. However, many gene therapy techniques
lack precision and may inadvertently insert an introduced piece of DNA into the wrong
part of the genome, interrupting a healthy and functioning gene.
36
what does crispr have the potential to do
h the potential to
increase crop productivity, eliminate genetic diseases, and better understand the purpose
of specific gene
37
how does the cas 9 system introduce genetic changes
induce genetic changes by
cutting DNA at a location specifically chosen by scientists, who make a synthetic sgRNA,
known as single guide RNA (sgRNA), to guide Cas9.
38
what is sgRNA made of
sgRNA is made from a single strand
of RNA rather than two RNA molecules like the gRNA within bacteria, yet the function
of sgRNA remains the same.
39
why does crispr using sgRNA cut the dna
This cutting of the DNA creates an opportunity for
nucleotides to be added, removed, or substituted into the selected sequence. In turn, this
can knockout, enhance, or change the function of a gene.
40
what are the 8 steps for using crispr for gene editing
1 Synthetic sgRNA is created in a lab that has a complementary spacer to the target DNA
that scientists wish to cut.
2 A Cas9 enzyme is obtained with an appropriate target PAM sequence.
3 Cas9 and sgRNA are added together in a mixture and bind together to create the
CRISPR-Cas9 complex.
4 The sgRNA-Cas9 mixture is then injected into a specific cell, such as a zygote.
5 The Cas9 finds the target PAM sequence and checks whether the sgRNA aligns with
the DNA.
6 Cas9 cuts the selected sequence of DNA.
7 The DNA has a blunt end cut that the cell will attempt to repair.
8 When repairing the DNA, the cell may introduce new nucleotides into the DNA at this
site. Scientists may inject particular nucleotide sequences into the cell with the hope
that it will ligate into the gap
41
uses of crispr in research
* Attaching a fluorescent protein to Cas9 to locate a specific gene in the genome
* Disrupting the expression of a gene to see the effect of that protein being knocked out. In turn, this helps
scientists identify the function of specific genes
42
uses of crispr in dealing w diseases
* Replacing a deleterious allele with a healthy allele
* Adding genes that code for proteins that decrease susceptibility to infectious diseases such as HIV/AIDS
* Modifying cancer-promoting genes to make them less influential
43
uses of crispr in agriculture
* Introducing pest and herbicide-resistance genes to increase the yield of crops
* Altering genes to promote increased growth rates to improve the yield of crops
44
limitations to crispr
To induce substitution mutations or knock-in a new segment of DNA, scientists must introduce the nucleotide sequence they wish to add into the cell and hope it is taken up by the DNA repair machinery.
This can be difficult to achieve with precision and is not consistently successful.
45
bioethical concept related to crispr
For example, the bioethical concept of non-maleficence discourages causing harm
wherever possible. According to this principle, an individual might oppose the law against
the gestation of genetically modified embryos out of concern for unforeseen negative
consequences on the pregnancy. On the other hand, imagine that a couple discovers their
fetus has a debilitating genetic condition that could be fixed by CRISPR-Cas9 technology.
The concept of non-maleficence could be used to argue for CRISPR-Cas9 in this case, as
the application of this technology could reduce the disadvantage or pain faced by the child or parents
46
other ethical concerns/approaches
* Safety – the possibility of off-target cleavages (edits in the wrong place) and mosaics
(some cells containing edited genomes, others not) mean that many scientists are
hesitant to use CRISPR outside of research.
* Informed consent – scientists cannot get consent from embryos to edit their genes.
If the embryo goes on to be born and one day has children of its own, these children
also will never have consented to scientists interfering with their genome.
* Inequality – there is concern that only wealthy people will be able to afford to use
CRISPR to treat genetic conditions or otherwise change their genes.
* Discrimination – CRISPR may be a threat to those who are judged by society as
biologically inferior, when in fact those individuals do not feel they need ‘fixing’ at all
47
what is the pcr chain
The polymerase chain reaction (PCR) is a DNA manipulation technique that amplifies
DNA by making multiple identical copies.
48
when is it used
It is used by scientists whenever there is an
insufficient amount of a DNA sample for testing. After undergoing the polymerase chain
reaction, scientists can run further analyses on the DNA such as: paternity testing
* forensic testing samples of bodily fluids
* analysing gene fragments for genetic diseases.
49
do scientists copy the entire genome during pcr
they focus on certain genes through the use of primers or
restriction endonucleases to make the process more efficient.
50
after each cycle of pcr what happens
After each cycle of the
polymerase chain reaction, the amount of DNA present is doubled
51
The polymerase chain reaction requires the following materials to take place:
The polymerase chain reaction requires the following materials to take place:
* a DNA sample that subsequently gets denatured and amplified through the polymerase
chain reaction
* Taq polymerase is required in the elongation stage to bind complementary nucleotides
to the single-stranded DNA
* nucleotide bases must be constantly available for Taq polymerase to create a new strand
that is complementary to the single-stranded DNA
* sequence-specific DNA primers join to the 3’ end of single-stranded DNA by
complementary base pairing to form the first segment of double-stranded DNA,
allowing Taq polymerase to attach and begin extending the DNA strand.
52
To begin the polymerase chain reaction...
a mixture of the above materials is placed into
a thermal cycler (Figure 1), where it undergoes the following processes:
1 Denaturation – DNA is heated to approximately 90–95 °C to break the hydrogen bonds
between the bases and separate the strands, forming single-stranded DNA.
2 Annealing – the single-stranded DNA is cooled to approximately 50–55 °C to allow
the primers to bind to complementary sequences on the single-stranded DNA.
3 Elongation – the DNA is heated again to 72 °C, which allows Taq polymerase to work
optimally. Taq polymerase binds to the primer, which acts as a starting point, and begins
synthesising a new complementary strand of DNA.
4 Repeat – the cycle (steps 1–3) is repeated multiple times to create more copies of DNA.
53
how many primers are needed in pcr and why
In the polymerase chain reaction, there are two different DNA primers needed. This is
because, during denaturation, the double-stranded DNA molecule has been separated
into two single-strands – the template strand and the coding strand.
Ultimately, having these two primers is necessary as the 5’ ends of both the template
and coding strands are different. As Taq polymerase only functions towards the 3’
end, a primer is needed for both strands to facilitate this directionality (Figure 3)
54
what do the forward and backward primers do
* the forward primer, which will bind to the start codon at the 3’ end of the template
strand. This causes Taq polymerase to synthesise a new DNA strand in the same
direction that RNA polymerase would function.
* the reverse primer, which will bind to the stop codon at the 3’ end of the coding strand.
This causes Taq polymerase to synthesise a new DNA strand in the reverse direction
that RNA polymerase would function.
55
Gel electrophoresis
Gel electrophoresis is a laboratory technique used by scientists to measure the size of
DNA fragments.
56
when is gel electrophoresis
It is typically used after a sample of DNA has been cut up using restriction
endonucleases or after a short sequence of DNA has been amplified using the polymerase
chain reaction.
57
The process of gel electrophoresis is described in the following steps (Figure 1): step 1
The process of gel electrophoresis is described in the following steps (Figure 1):
1 The DNA samples are placed in the wells at one end of the gel using a micropipette.
A standard ladder of DNA fragments with known sizes is also typically loaded into
one well. This is required for estimating the size of any unknown DNA fragments by
comparing them to the known fragments in the standard ladder. The gel is made of
agarose, a sponge-like jelly that is filled with tiny pores to allow movement of the DNA
fragments. This agarose gel is immersed in a buffer solution which helps carry an
electric current. 1 The DNA samples are placed in the wells at one end of the gel using a micropipette.
A standard ladder of DNA fragments with known sizes is also typically loaded into
one well. This is required for estimating the size of any unknown DNA fragments by
comparing them to the known fragments in the standard ladder. The gel is made of
agarose, a sponge-like jelly that is filled with tiny pores to allow movement of the DNA
fragments. This agarose gel is immersed in a buffer solution which helps carry an
electric current.
58
step 2 gel
2 An electric current is passed through the gel using two electrodes – one positive, one
negative. The negative electrode is positioned near the wells and the positive electrode
is at the opposite end of the gel. Since DNA is negatively charged due to the phosphate
backbone, it is attracted to the positive electrode. When the electrical current is applied,
DNA fragments will move from the wells, through the tiny pores in the agarose gel,
towards the positive electrode.
59
step 3 gel
3 Smaller DNA fragments move faster through the gel and so travel further than larger
fragments, which don’t move as easily through the pores in the agarose. After a few
hours, the current is switched off and the DNA fragments stop moving in the gel and
settle into bands. The DNA fragments are now separated based on size
60
step 4
4 DNA is difficult to see with the naked eye so the gel is stained with a fluorescent dye
such as ethidium bromide, allowing the bands of DNA to be visualised under an
ultraviolet (UV) lamp. This dye can be included in the gel before the experiment or
applied after.
61
gel electrophoresis separates dna based on what
When gel electrophoresis separates DNA fragments based on size, long fragments of DNA
collect in bands of DNA near the well, while shorter fragments form bands further from
the well.
62
what is a standard ladder + what does molecular size indicate?
A standard ladder contains a number of different DNA fragments with a known molecular
size. Molecular size indicates the length of a nucleic acid sequence and is measured in
base pairs (bp) or kilobases (kb).
63
why are standard ladders vital? what will influence the distance moved by DNA fragments? distance travelled in a gel can't be used to directly measure what?
Standard ladders are vital because DNA fragments of the same size don’t always travel
the same distance. Every gel type is different, and inconsistent experimental conditions
will influence the distance moved by DNA fragments. For example, in one gel a 100 bp
fragment may travel 7.8 cm whilst in another gel it may travel 8.6 cm, so distance travelled
in a gel can’t be used to directly measure molecular size.
64
what are the variations in the distance
These variations are due to
factors such as:
* voltage – the stronger the electric force generated by the electrodes the further DNA
travels towards the positive electrode
* gel composition – gels with a greater density and agarose concentration increase the
difficulty for larger fragments to move through
* buffer concentration – the greater the concentration of ions in the buffer the more the
electric current is conducted through the gel, which causes DNA to move further down
the lane
* time – the longer the electric current is applied, the further the DNA will travel.
Note: if too much time passes, the DNA may move out of the gel
65
how do genetic disorders occur, how small can these changes be?
Genetic disorders occur when individuals possess mutated alleles which prevent parts of
the body and its cells from functioning the way they should. These mutations can be as
small as a change of a single nucleotide.
66
what are str's and why are they used
STRs are small sections of repeated
nucleotides that vary in length between people and are found in the non-coding areas
of autosomal chromosomes. Because they are found in non-coding regions, they are not
affected by natural selection, and many hundreds of variant STRs can be found in the DNA
of each person due to their higher mutation rate than other areas of DNA. If the STRs in
two samples of DNA match, we can say with confidence that the two pieces of DNA belong
to the same person.
67
what else can dna profiling be used for
Using DNA profiling, we can also discover how related two people are. This is particularly
useful in parental testing when the identity of one of the parents is not known. Because
many of the STRs that we use are found exclusively on autosomal chromosomes, the child
must inherit half of their STRs from each one of their parents. In addition to identifying
criminals and parental testing, DNA profiling has been used to identify dead bodies, match
potential organ donors, and find lost relatives
68
how is a dna profile created through gel electrophoresis
One of the ways scientists construct a DNA profile is by using gel electrophoresis.
Scientists perform the polymerase chain reaction and run a gel focusing on particular
STRs, where each variant of an STR will separate according to size. If the individual is
heterozygous for an STR their gel will have two bands, whereas if they are homozygous
their gel will only have one thick band (Figure 7). In this way, scientists can accurately and
efficiently match two samples of DNA
69
what does gmo refer to
Any organism that has been altered using genetic engineering technologies is referred to
as a genetically modified organism (GMO).
70
what is a host organism
The organism that receives the altered gene/s
is referred to as the host organism, with the goal typically being to confer the host with a
desirable characteristic that was originally lacking from its genome.
71
2 types of gmo's
cisgenic
transgenic
72
cisgenic gmo
* Cisgenic organisms: a genetically modified organism that has genes from the
same species inserted into its genome. This process, known as cisgenesis, involves
transferring genes between organisms that could otherwise be bred together.
73
trangenic gmo
Transgenic organisms: a genetically modified organism that has genes from a different
species inserted into its genome. This process, known as transgenesis, results in an
organism that contains foreign DNA transplanted from a separate species.
74
Producing transgenic plants
Before considering each of these uses, let us take a brief look at how GMO technology is
used to create transgenic plants for agriculture. This process typically involves three stages
(Figure 4):
1 Gene identification: firstly, a particular gene of interest must be identified and
isolated. This gene of interest will usually be found in the genome of another species,
and have some important characteristics that would be useful for the host organism.
For example, the gene of interest may help with more efficient uptake of soil nutrients,
less reliance on fertilisers, and/or improved drought tolerance.
Last lesson, lesson 4E,
we explored bacterial
transformation and its uses
including producing insulin.
Transformed bacteria are
actually another example of
GMOs. By inserting a gene of
interest into a recombinant
plasmid which is then taken
up and expressed by a
transformed bacterium, we
have created a GMO (and
also a transgenic organism as
the gene of interest is from
another species).
219
2 Gene delivery: next, the isolated gene of interest must be delivered into the cells of
the host organism. This delivery may occur either via direct insertion of the DNA into
the genome of the plant itself or through the use of a bacterial plasmid that is able to
successfully transfer DNA between itself and the plant.
3 Gene expression: the transformed cell is then grown repeatedly (regenerated) using
plant tissue cultures under sterile conditions before being applied in the field for
agricultural use. The GM host organism is now able to express the new transgene as a
useful protein and can regenerate itself.
75
Use in agriculture: increasing crop productivity
One important use of GMO technology in agriculture is to increase crop productivity. In
other words, the agricultural industry uses GMO technology to increase its crop yield
(how much crop is produced) per unit of farming land. The quality of crops can also be
improved, for instance by increasing their nutritional value and their ability to grow in
different conditions.
76
Use in agriculture: increasing disease resistance
A second important use of GMOs in agriculture is to increase a crop’s resistance to disease.
By developing crops that are less impacted by harmful plant pathogens, scientists can
improve global food security by minimising crop destruction and the spreading of disease.
Furthermore, not only can a crop’s resistance to disease be increased, but so too can its
resistance to other damaging environmental factors, such as drought and herbivorous pests.
77
biological implications of gmo's pros
* GM crops usually have better crop productivity than non-GM crops. This means that more food can be grown using
less land, reducing habitat loss due to land clearing.
* Insect-resistant GM plants require fewer pesticides, which is better for the environment.
* GM foods can be made to have improved nutritional content, improving the health of individuals that consume them.
78
biological implications of gmo's cons
GM crops may lose their effectiveness if weeds or pests evolve resistance.
* Widespread use of GM crops could result in the loss of genetic diversity within crop populations.
* Cross-pollination between GM crops and wild species or weeds may cause genes to spread and lead to
unforeseen consequences
79
social implications of gmo's pros
* Increased crop productivity means more food can be produced, leading to better food security.
* Crops that are able to grow in more adverse conditions (e.g. drought-tolerant corn) protect against famine,
improving food security.
* Herbicide-tolerant crops reduce labour demands as farmers don’t need to pull weeds by hand, instead spraying
chemicals that selectively kill weeds but not crops.
* Increased crop yields result in larger profits for farmers.
* GM foods can be made to have improved flavour and texture, giving consumers a more appealing product.
* GM foods can be made to have improved nutritional content. This leads to a reduction in nutritional deficiencies,
creating healthier populations
80
social implications of gmo's cons
* Having to buy new seeds each season may be costly for farmers.
* Complex legal issues surrounding the use of GM products may cause farmers undue stress and anxiety related
to regulation.
* There are strict packaging and marketing regulations for GMO producers that may not be complied with if either
the producer or consumer are undereducated on these regulations
81
ethical implications of gmo's pros
* Some people believe that using genetic modification is an ethical imperative given the potential for widespread
benefits, including nutrition, wealth, and the overall health of humanity, especially in developing nations.
82
ethical implications of gmo's cons
Some people consider GMOs to be unnatural, or like we are ‘playing God’.
* Some people believe that GM foods are unsafe to eat and choose not to eat them as a result. This is especially true
if there is lack of long-term evidence of healthy use.
* Some people believe that genetically modifying animals for human benefit is inhumane – many anti-animal GMO
arguments apply to animal agriculture in general.
* The fact that companies can own the rights to GM crops is considered by some to be unethical due to companies
possibly making unfair demands of farmers. This ownership power divide can materialise in a range of ways,
including the following:
–Cross-pollination of non-GM crops by nearby GM crops could result in the non-GM farmer being sued by the
patent-owner.
–Farmers can’t reuse seeds from some GM crops and must buy new expensive seed supplies each year from
biotechnology companies
