Chapter 4 - DNA manipulation Flashcards
1
Q
agarose gel
A
a sponge-like gel used
in gel electrophoresis that contains
pores for DNA fragments to move
through
2
Q
amplify
A
to increase the quantity of a molecule by making many copies
3
Q
anneal
A
the joining of two
molecules, for example two
complementary DNA strands
during the cooling phase of PCR
4
Q
antibiotic resistance gene
A
gene
which confers antibiotic resistance
5
Q
bacterial transformation
A
the
process by which bacteria take
up foreign DNA from their
environment. Scientists use this
process to introduce recombinant
plasmids into bacteria
6
Q
bacteriophage
A
a virus that infects
prokaryotic organisms
7
Q
band
A
a line seen in the gel after
running gel electrophoresis that
corresponds to a collection of DNA
fragments of a specific size
8
Q
base pair (bp)
A
a unit of
measurement that corresponds to
one nucleotide
9
Q
blunt end
A
the result of a straight cut across the double-stranded DNA by an endonuclease resulting in no overhanging nucleotides
10
Q
buffer
A
an ion-rich solution that
carries electrical current through
the agarose gel
11
Q
cisgenic organisms
A
a genetically
modified organism that contains
foreign genetic material from
a sexually compatible donor
organism, typically from the
same species
12
Q
CRISPR-associated protein 9
(Cas9)
A
an endonuclease that
creates a blunt end cut at a site
specified by guide RNA (gRNA)
13
Q
CRISPR-Cas9
A
a complex formed
between gRNA and Cas9 which
can cut a target sequence of
DNA. Bacteria use this complex
for protection from viruses and
scientists have modified it to
edit genomes
14
Q
CRISPR
A
short, clustered repeats
of DNA found in prokaryotes
which protect them against
viral invasion
15
Q
deleterious mutation
A
a change in DNA that negatively affects an individual
16
Q
denature
A
the disruption of
a molecule’s structure by an
external factor such as heat
17
Q
diabetes
A
a disease where the
body cannot properly produce
or respond to insulin
18
Q
differentiation
A
the process in which cells develop specialised characteristics, typically transforming them from one cell type to another more specialised cell type
19
Q
DNA profiling
A
the process of identification on the basis of an individual’s genetic information
20
Q
electrode
A
conductors of electricity
that are attached to both ends of a
gel allowing an electrical current to
pass through it
21
Q
electroporation
A
a method that involves delivering an electric shock to bacterial membranes to increase their membrane permeability and increase the likelihood of bacterial
transformation
22
Q
elongate
A
to synthesise
a longer polynucleotide
23
Q
embryo
A
an early stage of
development in an organism.
In humans, used to refer to the
organism during the first eight
weeks of development
24
Q
endonuclease
A
an enzyme that breaks/cleaves the phosphodiester bond between two nucleotides in a polynucleotide chain (‘restriction endonuclease digestion’.)
25
ethidium bromide
a fluorescent
dye that binds to DNA fragments in
a gel and allows them to be easily
visualised under ultraviolet light
26
forward primer
a DNA primer
that binds to the 3’ end of the
template strand and reads the
DNA in the same direction as
RNA polymerase
27
fusion protein
a protein made
when separate genes have been
joined and are transcribed and
translated together
28
gel electrophoresis
a technique
that separates DNA fragments
based on their molecular size
29
gene knock-in
a technique in gene
editing where scientists substitute
or add nucleotides in a gene
30
gene knockout
a technique in
gene editing where scientists
prevent the expression of a target
gene to understand its function in
an organism
31
gene of interest
a gene scientists want to be expressed in recombinant bacteria. This gene often encodes a protein we wish to produce in commercial quantities. Also known as the desired gene
32
gene therapy
repairing genetic mutations by replacing a defective gene with a healthy one
33
genetic engineering technologies
refers to the artificial alteration
of an organism’s genome via
the exchange of foreign genetic
material, typically from another
organism. This is often done
external to the organism via the
use of a transfer vector such as a
plasmid. Also known as genetic
recombination technologies
34
genetic engineering
the process
of using biotechnology to alter the
genome of an organism, typically
with the goal of conferring some
desirable trait
35
genetic modification
the
manipulation of an organism’s
genetic material using
biotechnology
36
genetic modification
the manipulation of an organism’s genetic material using biotechnology
37
genetic testing
screening an individual’s DNA for anomalies that may make them susceptible to a particular disease or disorder
38
genetically modified organism
(GMO)
an organism with
genetic material that has been
altered using genetic engineering
technology
39
guide RNA (gRNA)
RNA which has a specific
sequence determined by CRISPR
to guide Cas9 to a specific site
40
heat shock
a method that involves
rapidly increasing and decreasing
the temperature to increase
membrane permeability in order to
enhance the likelihood of bacterial
transformation
41
heterozygous
having different
alleles for the same gene on
homologous chromosomes
42
homozygous
having identical
alleles for the same gene on
homologous chromosomes
43
host organism
the organism
which researchers wish to
genetically modify
44
insulin
a hormone secreted by the pancreas to control blood glucose levels
45
kilobase (kb)
a unit of
measurement that corresponds
to one thousand nucleotides.
May also be written as kbp
46
lane
the column of the gel corresponding to each sample of DNA
47
ligase
an enzyme that joins
molecules, including DNA or
RNA, together by catalysing the
formation of phosphodiester bonds
48
origin of replication (ORI)
a
sequence found in prokaryotes
that signals the start site of DNA
replication
49
overhanging nucleotides
unbonded nucleotides on the ends
of the DNA strand resulting from a
staggered cut
50
plant tissue culture
a range of
techniques used to grow plant
cells, tissues, or organs under
sterile conditions using a nutrient
culture medium, such as an agar
plate or nutrient broth of known
composition. It is widely used to
produce clones of a plant
51
plasmid vector
a piece of circular DNA that is modified to be an ideal vector for bacterial transformation experiments (contains: - restriction endonuclease sites - antibiotic resistance genes - origin of replication - reporter gene)
52
plasmid
a small, circular loop
of DNA separate from the
chromosome, typically found
in bacteria
53
polymerase
an enzyme that
synthesises a polymer from
monomers, such as forming
a DNA strand from nucleic acids
54
polymerase chain reaction (PCR)
a laboratory technique used
to produce many identical copies
of DNA from a small initial sample
55
primer
a short, single strand
of nucleic acids that acts as a
starting point for polymerase
enzymes to attach
56
primer
a short, single strand
of nucleic acids that acts as a
starting point for polymerase
enzymes to attach
57
protospacer
a short sequence
of DNA extracted from a
bacteriophage by Cas1 and Cas2,
which has yet to be incorporated
into the CRISPR gene
58
protospacer adjacent motif (PAM)
a sequence of two-six nucleotides
that is found immediately next to
the DNA targeted by Cas9
59
recognition site
a specific target
sequence of DNA upon which
restriction endonucleases act - generally 4-6 palindromic (5'-3' same on each strand) nucleotides
60
recombinant plasmid
a circular DNA vector that is ligated to incorporate a gene of interest
61
reporter gene
gene with an easily
identifiable phenotype that can be
used to identify whether a plasmid
has taken up the gene of interest
62
restriction endonuclease
any enzyme that acts like molecular scissors to cut nucleic acid strands at specific recognition sites. Also known as a restriction enzyme
63
reverse primer
a DNA primer
that binds to the 3’ end of the
coding strand and reads the
DNA in the reverse direction
to RNA polymerase
64
short tandem repeats (STR)
short, repeated sequences of
nucleotides found in the noncoding regions of nuclear DNA
65
silenced
describes a gene that is
prevented from being expressed
66
single guide RNA (sgRNA)
guide RNA utilised by scientists to
instruct Cas9 to cut a specific site
when using CRISPR-Cas9 in gene
editing
67
spacer
short sequences of
DNA obtained from invading
bacteriophages that are added into
the CRISPR sequence
68
standard ladder
a mixture of DNA
fragments of known length that are
used to infer the size of fragments
in a sample
69
sticky end
the result of a staggered cut through double-stranded DNA by an endonuclease resulting in overhanging nucleotides
70
Taq polymerase
a heat-resistant
DNA polymerase enzyme isolated
from the bacteria Thermus
aquaticus, which amplifies a
single-stranded DNA molecule
by attaching complementary
nucleotides
71
thermal cycler
a laboratory
apparatus which alters the
temperature in pre-programmed
steps for temperature-sensitive
reactions like PCR
72
transgene
a gene that has been
artificially introduced into the
genome of a separate organism
(usually of another species)
73
transgenic organism
a genetically
modified organism that contains
foreign genetic material from a
separate species (or recombinant
DNA from the same species that
has been manipulated before
introduction)
74
vector
a means of introducing
foreign DNA into an organism.
Plasmids are a popular vector in
bacterial transformation
75
virus
a non-cellular, infectious
agent composed of genetic
material enclosed in a protein coat
that requires a host cell to multiply
76
well
an indent in the gel into
which a DNA sample is loaded
77
zygote
the diploid cell formed by the combination of two haploid gamete cells
78
Steps of a bacterium fighting a bacteriophage with CRISPR-Cas9
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
EXPRESSSION - 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. gRNA forms a hairpin loop-like structure from the transcribed palindromic repeats either side of the spacer.
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
79
To use CRISPR-Cas9 for gene editing, the following steps must be taken:
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.
80
Materials needed for polymerase chain reaction:
* 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.
81
Steps of polymerase chain reaction
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.
82
process of gel electrophoresis
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
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.
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.
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.
83
Steps in producing insulin
Creating the recombinant plasmid
1 Plasmid vectors were prepared which contained the ampR gene to encode for
antibiotic resistance to ampicillin and tetR to encode for antibiotic resistance to
tetracycline. tetR acted as a reporter gene and had a specific recognition site to one of
the restriction endonucleases used in step 2 inside it.
2 Two plasmid vectors were used - one for insulin subunit A and one for insulin
subunit B. Using the two restriction endonucleases EcoRI and BamHI, both plasmid
samples, the insulin A subunit gene, and the insulin B subunit gene, were all cut to
form complementary sticky ends. It is once again important to note that the inserted
insulin subunit genes were without introns as they were produced in a laboratory.
In addition, the insulin subunit genes were also created with an extra codon coding
for methionine at the beginning of the insulin gene. DNA ligase was then used to
re-establish the sugar-phosphate backbone and create two different recombinant plasmids
Creating transformed bacteria
3 The plasmids were added to a solution of E. coli bacteria and some of the recombinant
plasmids were taken up by the bacteria.
4 To determine which bacteria successfully took up plasmids, the bacteria cultures were
spread and incubated onto agar plates containing the antibiotic ampicillin. Colonies
that formed were identified to have taken up a plasmid, however, to determine which
colonies contained bacteria that took up recombinant plasmids (as opposed to taking
up non-recombinant plasmids which were also resistant to ampicillin), another test
with tetracycline was needed. Some of each of the colonies from the ampicillin plate
were spread onto agar plates containing tetracycline. If the bacteria were not resistant
to tetracycline, then it was known that they contained recombinant plasmids. This is
because the insertion of the insulin subunit gene interrupted the tetR gene, thus making
bacteria with recombinant plasmids susceptible to tetracycline. These plasmids were
then collected.
5 The plasmids were then cut open once again using EcoRI, to insert another gene
called lacZ (minus its stop codon). lacZ produces ß-galactosidase, a large enzyme.
When expressed, recombinant plasmids will produce an insulin subunit protein that
is attached to the much larger ß-galactosidase enzyme, in what is known as a fusion
protein. This is important as it protects the smaller insulin subunit protein from
digestive enzymes within E. coli that may destroy it.
6 The recombinant plasmids containing lacZ were added to a new solution of E. coli
bacteria and some of these new recombinant plasmids were taken up by the bacteria.
7 To determine which bacteria successfully took up the new recombinant plasmids, a
test was performed that relied on the function of ß-galactosidase. The ß-galactosidase
enzyme was known to convert a compound called X-gal, which results in a compound
changing from colourless to being blue coloured. Consequently, the E. coli were plated
on agar plates containing ampicillin and X-gal. Colonies that grew and were blue in
colour were identified as containing recombinant plasmids due to the presence of
ß-galactosidase. These bacteria were capable of producing the insulin subunit proteins
that were attached to ß-galactosidase.
Protein production and extraction
8 Transformed bacteria that contained the recombinant plasmid were then placed into
conditions to exponentially reproduce before their membranes were broken down, and
the insulin subunit and ß-galactosidase fusion proteins were extracted.
The compound cyanogen bromide was added to break down the methionine that was
added at the start of the insulin gene. In doing so, it separated the insulin subunit from
the ß-galactosidase, allowing for the isolation and purification of the insulin subunit.
9 The two insulin chains were then mixed together, which allowed the connecting
disulphide bonds to form and create functional human insulin.
