Experimental Strategies Flashcards
(87 cards)
1
Q
Recombinant DNA technology
amm the seqs
A
a set of techniques for amplifying, maintaining, and manipulating DNA sequences in vitro and in vivo
2
Q
Recombinant DNA technology - applications
A
- Fragment DNA into easily managed pieces and purify them
- Replicate DNA fragments of interest in vitro
- Combine DNA fragments to construct recombinant DNA molecules
- Determine sequence of specific DNA molecules
- Identify fragments of DNA containing complementary sequences
- Introduce specific DNA molecules into living organisms
- Assay effects of introduced DNA into organisms
3
Q
Nucleases
Definition and first discovery
A
- Nuclease enzymes that recognize specific DNA sequences and
cuts both DNA strands at this site - First identified in bacterial cells as an immune system against
viruses
4
Q
Restriction enzymes
Resulting ends
A
- Some restriction enzymes (ex nucleases) make cuts called sticky ends, meaning single stranded overhangs are left after the cut
- Have recognition sequences on these overhangs
- Two fragments with sticky ends can combine through complementary base pairing
5
Q
DNA digest
Treating DNA with
A
treating DNA with restriction enzymes
different restriction enzymes have different recognition sequences
6
Q
Estimating cut size frequency
A
one cut every 4^n nucleotides
n = number of nucleotides in recognition sequence
1/4^n = probability of finding an identical sequence by chance
7
Q
Restriction enzyme examples and their cut size/frequency
A
EcoRI
cut every 4^6 = 4096 nucleotides (1/4096)
Recognition seq: 5’-GAATTC-3’
AluI
cut every 4^4 = 256 nucleotides (1/256)
Rec seq: 5’-AGCT-3’
8
Q
Cut and fragment relationship
A
n fragments
n-1 cuts
ex. 2 fragments = 1 cut
circular plasmid, # cuts = # fragments
9
Q
Restriction mapping of lambda phage
A
- map restriction enzyme sites
(before sequencing was available) - expose DNA to various restriction enzymes and analyze fragment sizes using gel electrophoresis
- smaller fragments migrate further
10
Q
Molecular cloning
A
genomic DNA is digested with restriction enzymes
- sequence of interest is isolated using specific restriction enzymes that cut near the gene of interest
- Isolated fragments can be inserted into a vector (e.g. plasmid) and introduced into a biological system that will amplify the DNA
(e.g E. coli)
11
Q
DNA clones
General definition
A
identical copies of the replicated DNA fragment
12
Q
3 steps in molecular cloning
A
- Combine vector and DNA fragment of interest using DNA ligase to produce a recombinant DNA clone
- Insert plasmid into biological system (e.g E. coli)
- Allow recombinant plasmid to replicate in biological system
ex. digest plasmid vectors and human DNA with EcoRI, combine fragments so sticky ends attach, use ligase to attach
13
Q
Transformation; to insert recombinant plasmid into E coli
A
Protocol
* Generating competent cells: E. coli treated with chemicals that creates
pores in their cellular membrane
* Keeping competent cells on ice
* Exposing cells to heat shock
* Immediately returning cells to ice
* Allowing cells to recover in nutrient media
plasmid is inserted during heat shock
14
Q
Selecting transformant cells
(cells that have successfully taken up the recombinant plasmid)
A
Plasmids normally contain a gene for resistance to antibiotics like ampicillin (ex. beta-lactamase gene)
- After cells recover from heat shock, plate the E. coli cells on petri dishes with ampicillin
- Each colony that grows represents a single cell that continued to asexually reproduce
- Select a colony and inoculate cells in fresh media
- As the cells replicate, the DNA fragment of interest also replicates
TLDR; if they can grow in ampicillin, they have the plasmid
15
Q
Verifying Presence of DNA Insert within Plasmid;
Blue-White Screening
A
- Some plasmids have lacZ gene with
restriction cut sites inside that gene - If DNA fragment of interest gets successfully
ligated into plasmid, lacZ gene will get a
frameshift mutation, inactivating the gene - Plate cells on petri dish with X-gal, an analog
of lactose - X-gal, when broken down by beta-
galactosidase (lacZ product) produces blue
dye - Blue colonies indicate the insert is missing in
plasmid - White colonies indicate the insert is present (bc mutates the lacZ)
16
Q
Multiple cloning site (MCS)
And adjacent to
A
- place with lots of restriction enzyme cuts
- where you are going to place your gene insert
- adjacent to a promoter that will promote gene of interest
17
Q
Verifying Presence of Insert within Plasmid; by size
A
- Select a bunch of colonies of transformed cells
- Grow them in separate cultures
- Extract plasmid DNA
- Digest plasmid DNA with a restriction enzyme that cuts within insert DNA fragment
- Inspect fragment sizes through gel electrophoresis
- Typically recombinant plasmids increase in size from non recombinant plasmids (in bp)
18
Q
Can all insert genes be properly expressed in E. coli?
+ solution
A
NO
* E.g genes that produce protein with a number of post-translational
modifications, like from eukaryotes
Eukaryotic expression vectors could be used instead in eukaryotic cells
* Insertion of plasmid or other vector into yeast or tissue culture cells
* These vectors have the regulatory sequences suitable for eukaryotic systems
19
Q
Production of Human Insulin in E. coli
historical application
A
- A gene encoding human insulin was among the first human genes to be expressed in E. coli
- Pancreatic cells initially synthesize a 110–amino acid protein called preproinsulin that is not secreted and does not function until processed
- The first cleavage removes the 24 N-terminal amino acids to
produce proinsulin; next 35 additional amino acids are removed - After the first two cleavage events, additional cleavage
generates two amino acid chains, the A and B chains, which
are joined by disulfide bonds to produce insulin - The amino acid sequence of insulin was determined in the 1950s
- Chemically synthesized DNA encoding the two chains
separately was used to express insulin in E. coli before the gene was identified (in the 1970s)
20
Q
Direction of DNA replication / transcription
A
5’ to 3’
N to C
21
Q
The Two-Chain Method of Expression
To make insulin
A
- Each synthetic gene (for the A and B chains) was cloned into a
separate plasmid vector, fused in frame to the 3′ end of the lacZ
gene - Constructs consisting of two or more gene segments joined
together are called fusion genes; the protein products of these are called fusion proteins
so lacZ also starts the insulin gene
- Extra codons for methionine were introduced at the site between the lacZ gene and each insulin gene
TLDR; plasmid containing the beta galactosidase gene with the insulin B chain just slightly downstream and a stop codon between them
22
Q
The Two-Chain Method of Expression
removing the beta galactosidase portion
A
- Cyanogen bromide treatment of fusion proteins cleaves the C-terminal end of methionine; this is used to separate the components of each fusion protein
- Gene transcription is induced by lactose in the absence of glucose
- After cleavage with cyanogen bromide, the A and B chains are purified from the host strains and mixed together under conditions that allow disulfide bridge formation
23
Q
Generation of transgenic animals
3 conditions
A
Injecting vectors (e.g plasmid) or mRNA or protein into embryo
with the goal of having foreign DNA inserted into genome
- Embryo must be at the right stage of development and survive microinjection
- Transgene must be incorporated into germline cells
- Transgene must be passed on to offspring after mating transgenic individual
24
Q
Cloning: Dolly the sheep
A
- First animal cloned in 1996
- Nucleus isolated from adult somatic cell (mammary gland cell)
- Nucleus injected into an egg (oocyte) with its nucleus removed
- Cell was then shocked, stimulating cell division and development into embryo
25
Is Dolly the sheep an exact clone of her nucleus donor
no
mitochondrial DNA from her surrogate
epigenetic impacts from surrogate
= genotypically and phenotypically similar
26
Model organisms
organisms with:
- short generation times
- easy manipulation in a laboratory
27
E Coli
| Chromos
Gen time
Genome size
Protein coding genes #
# chromosomes: 1 (+plasmids)
gen time: 20 mins
genome size: 4.64 Mb
# chromos: 1 (+ plasmids)
# protein coding genes: 4262
- best studied prokaryote
- one of the easiest life forms to maintain in the laboratory
- has advanced our understanding of mechanisms behind DNA replication, transcription, and translation
28
S cerevisiae
| Chromos
Gen time
Genome size
Protein coding genes #
# protein coding genes:
baker's yeast
gen time: 2-3 hours
genome size: 12.2 Mb
# chromos: 16 (2n=32)
# protein coding genes: 6728
- eukaryotic cell with compartmentalized organelles
- can be cultivated as easily as E coli
- used for baking and fermenting alcohol
29
C elegans
| Chromos
Gen time
Genome size
Protein coding genes #
Neurons
# chromos: 5 (2n = 10)
nematode worm
gen time: 3 days
genome size: 103 Mb
# protein coding genes: 20 452
- one of the simplest multicellular life forms
- first animal to have its nervous system (connectome) mapped
~ 300 neurons
30
D melanogaster
| Chromos
Gen time
Genome size
Protein coding genes #
Neurons
# chromos: 4 (2n = 8)
fruit/vinegar fly
gen time: 2 weeks
genome size: 169 Mb
# protein coding genes: 14 217
- greatest model organism for genetics
- related to autosomal and sex-linked inheritance, genetic mapping, and more
- neuronal circuits involved in courtship, aggression, feeding, memory/learning, sensory systems, etc.
~ 100 000 - 200 000 neurons
31
M musculus
| Chromos
Gen time
Genome size
Protein coding genes #
Neurons
# chromos: 20 (2n=40)
house mouse
gen time: 10 weeks
genome size: 2731 Mb
# protein coding genes: 22 322
- best studied mammal
- many tools for genetic and neurological manipulation (like drosophila)
- closest relative to humans of the model organisms
~ 71 000 000 neurons
32
Choosing a model organism
pick the simplest possible
cellular processes = e coli or s cerevisiae
physiological patterns for neuronal circuits for simple behaviours = c elegans
short generation time and complex multicellular = drosophila
similar to humans and complex brain system = mice
33
Forward genetics
the identification of genes by mutant genotypes
- breed individuals exposed to to mutagens to wild-type individuals
- screen offspring for mutations through selective breeding
ex. the discovery of the white gene in drosophila
34
F1 screen (mutagenesis strategies)
* expose males to mutagen (no recombination) and mate them to wild-type females
* dominant mutations appear in F1 progeny
* can collect multiple mutants and generate pure-breeding stock
dominant mutations are more rare
35
F3 screen (mutagenesis strategies)
* expose males to mutagen and mate them to wild-type females
* isolate multiple F1 progeny and mate them to wild-type individuals
* collect and interbreed F2 progeny
* identify recessive mutants in F3 generation and collect multiple to begin a pure-breeding stock
recessive mutations are more common
36
F2 screen (mutagenesis strategies)
* mutagenize germ-line progenitors
* allow F1 individuals to self-fertilize
* newly induced mutations should be present in both male and female gametes
* identify recessive mutations in F2 individuals
limited to some specific model organisms
(not drosophila)
- good for plants
37
X-linked mutations
attached-X
* in drosophila, attached-x mutants are a great tool if an x-linked mutant is generated
* attached-x are phenotypically female, X chromos inherited together
* x-linked male mutants directly pass their X chromosome to male offspring
sex depends on ratio autosomal : x
X^X Y = mother
Xm Y = father
offspring:
X^X Y = females
Xm Y = males
X^X Xm = dead females
Y Y = dead males
38
Determining number of mutated genes in forward screen
complementation test
* If crossing two recessive mutants produces wild-type progeny, there are
two separate genes which are mutated
* If crossing two recessive mutants produces mutant progeny, there is only
a single mutated gene
39
Reverse genetics
Begin with a gene of interest and manipulate the gene to identify mutant phenotypes
opposite of forward genetics
used more in modern times bc genomic sequences known
40
Reverse genetics approaches (4)
- mutagenesis through gene editing
- gene knockout
- gene knockdown
- reporter genes (like GFPs)
41
CRISPR-Cas9 gene editing
Cas 9 = nuclease that makes double-stranded breaks
requires (one or multiple) guide RNA(s) that is complementary to the genomic target
cuts repaired through NHEJ or SDSA
Inject:
* engineered guide RNA
* Cas9 mRNA
* donor DNA template
42
Gene knockout
* deletion of the coding region of a gene - can be done through CRISPR
* induce DSB at the target gene
* supply a template containing a selectable marker that alters phenotype of the organism
selectable marker replaces deleted coding sequence and allows confirmation of knockout (ex. GFP)
43
Gene knockdown
* RNAi can be used to silence a gene of interest
* insert gene that produces RNA that can complementary base pair with mRNA of interest
* if this inserted gene is expressed in high amounts, a gene of interest will be silenced through degradation of mRNA
44
Reporter genes
| Definition and 2 examples
genes whose protein products can be directly or indirectly detected
ex. green fluorescent protein (GFP)
ex. lacZ gene (beta galactosidase)
- degrades X-gal (chemical that mimics lactose), produces blue when degraded
- used in microscopic life forms
45
Two types of fusion genes
(Reporter genes are often fusion genes)
Normal gene
5' upstream regulator / coding sequence / 3' downstream regulator
Transcriptional fusion
- replace coding sequence of original with reporter
- presence of GFP indicates where gene is transcribed and translated
= reporter phenotype
Translational fusion
- put reporter after the coding sequence
- GFP tagged onto translated protein to indicate where the protein ends up
= mixed phenotype
46
Enhancer trapping
the process of inserting a transgene with a weak promoter in a genome
promoter to weak to express the transgene at significant amounts
if gene inserted near an endogenous enhancer, the gene could be expressed at high amounts and with specific expression patterns
47
GAL4-UAS system
| Comes from, used for
yeast transcriptional regulation
many drosophila have engineered genes under control of these sequences
der)
48
Why do flies use the GAL4-UAS system
* UAS specific to GAL4 protein
* flies have no endogenous transcriptional activators that bind to UAS
and
they have no endogenous expression of GAL4 protein in their genome bc it is derived from yeast
= enables a system where GAL4 expression can be controlled by a specific promoter (driver) and a gene of interest can be inserted under the regulation of UAS (responder)
49
When is the GAL4-UAS system active
only when a driver line fly is crossed to a responder line fly
(lines don't show transgene expression alone)
promoter - GAL4 -- UAS - GFP (or any gene)
50
Driver line
GAL4 sequence fused with a promoter sequence
can force GAL4 expression in specific tissues (specific organs, neurons, localized cells, etc)
51
Responder line
UAS sequence fused with a target gene sequence
52
In what way is the GAL4-UAS system like a swiss army knife
thousand of different driver and responder lines
can be mixed and matched
custom ones can be made via CRISPR
53
5 common applications of GAL4-UAS
| 4 systems + 1 manipulation
1. Reporter system: to monitor where a gene of interest is expressed
2. Gene knockdown system: by using GAL4 to express RNAi
construct
3. Over-expression system: by using GAL4 to express an
endogenous gene at higher amounts
4. Rescue system: by using GAL4 to express a gene in knockout
mutants to determine if wild-type phenotype is rescued
5. Manipulate neurons: by using GAL4 to express proteins that
activate or silence neurons, in specific tissues
54
GAL4-UAS as a reporter system
+ use
driver line parent
= promoter-GAL4 (homo)
responder line parent
= UAS-GFP (homo)
F1
promoter-GAL4 / UAS-GFP
can use any promoter fused to GAL4
useful for finding where a gene of interest is expressed
55
Two types of promoters
1. specific
- ex. promoter of a specific neuron type
- leads to GFP expression only in a specific neuron
2. general
- ex. promoter of actin gene
- leads to GFP expression in every fly cell
56
GAL4-UAS and RNAi
driver line parent
= actin-GAL4
responder line parent
= UAS-RNAi construct
* All offspring with driver and responder will have the RNAi construct expressed in all cells
* RNAi construct will bind to specific mRNA of interest, preventing protein synthesis
* Can also be more restricted by controlling GAL4 through a different promoter (e.g. expressing
RNAi construct in specific neurons)
57
Over-expression
* Offspring with driver and responder will express gene of interest
in all cells in addition to wherever that gene is endogenously expressed
* Doubles gene expression
* Can express gene in specific tissue using a more restrictive promoter (e.g in certain neurons, in specific muscle tissue, reproductive tissue, etc.)
58
Forcing the expression of a Gene in Knockouts
(Rescue)
Restores expression of a gene that was knocked out
...
59
GAL80
* Natural repressor of GAL4
* Can be used as a separate driver to inhibit GAL4 in specific tissues
* Example, express GAL4 protein in all neuron tissues and express GAL80 gene in neurons
within optic lobe
* Result is inhibition of GAL4 only in visual neuron
60
GAL80^TS
* Temperature sensitive GAL80
* Enables temporal activation of GAL4 by simply heat shocking flies
* Example, keep GAL4 inactive during a fly’s development, but activate GAL4 during an
experiment
At restrictive temperature (cooler), GAL80^TS inhibits GAL4, preventing gene expression
At permissive temperature (warmer), GAL80^TS is unstable, allowing gene expression
61
GAL4-UAS tetanus
* GAL4-UAS system can be used to express tetanus toxin in
specific neurons
* This toxin silences neurons from firing
62
GAL4-UAS optogenics
| In neurons, inserting a receptor
- system used to activate neurons
- involves forcing the expression of a light-sensitive receptor in
neurons such as channel rhodopsin 2 (ChR2)
- this excites neurons in the presence of blue light
63
Argininemia (ARG)
- autosomal recessive condition
- caused by a deficiency of the enzyme arginase - breaks down arginine
- ammonia can build up in the bodies of those with ARG
= poor growth, poor muscle control, significant learning delays
64
Patient BK and ARG treatment
- parents did testing shortly after birth
- found out about condition
- gave appropriate treatment plan
= relatively normal life + hitting milestones
- found out that BK's parents were both heterozygous carriers = risk to future children
- treatment is low protein diet, ammonia clearing medication, regular blood testing
65
Medical genetics
aims to diagnose and manage medical, physiological, and social aspects of hereditary disease
collaborative field between physicians, diagnostic technicians, laboratory researchers, genetic counsellors,
etc
66
3 primary goals of medical genetics
1. Diagnose hereditary conditions in infants
2. Provide the treatments and care for infants with a rare inherited condition
3. Gather information from the patient and family to address
whether future children are at risk of being born with condition
67
Three categories of hereditary diseases
1. Mendelian conditions
* autosomal dom
* autosomal recess
* x-linked dom
* x-linked recess
2. Chromosomal conditions
* non-disjunctions
* chromosomal translocations
* chromosomal inversions
3. Multifactorial conditions
* influence of multiple genes and environmental factors
* diabetes, heart disease, cancer
68
The goal of medical geneticists
to diagnose which category a "disease phenotype" falls into
69
Online Mendelian Inheritance of Man (OMIM)
- tool for a medical geneticist
- open source database
- has a wealth of genetic info for human phenotypes, including disease
- great source for quick facts, and references to primary literature for further study
- also good for location of non-disease phenotypes
- great resource for researching genes that have been identified in traits known to vary in humans
ex. chromosomal location, gene function, documented mutations, phenotypes
70
Genetic location
#L###
ex. ARG is at 6q232
on chromosome 6
location q232
71
Pedigree analysis
used by genetic counsellors before genetic tests are done
* The various pedigree problems you have been challenged to solve this semester represents something practical genetic counsellors do
* Physicians may refer parents to a genetic counsellor if they suspect a genetic disease runs in their
families
* Can be informative to couples that are starting a family
72
2 types of genetic screening
1. newborn genetic screening
2. prenatal genetic screening
* non-invasive protocols
* invasive protocols
73
Newborn genetic screening
* Standard protocol in many countries
* Most likely all of us got newborn genetic tests
* Involves a “heel-prick” where a blood sample is taken
* Blood sample used for checking baby’s physiology and genetic screen
74
Phenylketonuria (PKU)
Phenylketonuria was the condition that lead to the development of newborn screening
* PKU is an autosomal recessive condition
* Caused by absence of enzyme phenylalanine hydroxylase which converts phenylalanine (Phe) to tyrosine
* PKU results from buildup of Phe which is toxic to the nervous system
* Children born with PKU appear normal at birth, symptoms appear
within a few months after birth
* Symptoms include severe mental and developmental impairment
75
Living with PKU (treatment)
PKU easily treated with strict dietary adjustments
* Low protein diet
* Avoiding artificial sweeteners – especially
aspartame
* It is estimated since the 1960s, more than
50 000 babies born with PKU globally have gone on to have normal lives
76
Prenatal genetic screens
| definition + when to use invasive vs noninvasive
Genotyping a child before they are born
* There are various protocols that are invasive and may involve a small risk of harming the fetus, while others are non-invasive
* Invasive methods may be encouraged by physician if non-invasive
methods are inconclusive and if there are concerns about child inheriting a condition that runs in the family
77
Invasive prenatal tests
* Goal is to extract a small sample of stem cells from amniotic fluid or chorion
* Cells can be cultured and genetic tests can be done on cultured cells
* Tests can include examining chromosome karyotype, DNA genotyping, etc
1. Amniocentesis
- syringe through abdomen takes amniotic fluid with stem cells
2. Chorionic villus sampling (CVS)
- catheter through birth control to chorion = membrane attached to amniotic fluid
78
Noninvasive prenatal tests: Ultrasound imaging
* Very common
* Can detect conditions such as Down syndrome (webbed neck)
* Can detect neural tube defects by examining head and spine of fetus
* May not always be conclusive
79
Noninvasive prenatal tests: Fetal cell sorting
* Fetal cells may enter into mother’s blood circulation in low amounts
* This process identifies and isolates fetal cells from blood samples taken from mother
* This technique has been used with some success, but requires further development and innovations to be more reliable
80
Karyotype used to detect chromosomal abnormalities
Robertsonian translocation
- chromosome 21 translocates to chromosome 14
= more likely to have a child with trisomy 21 bc chromo 14 comes with an extra chromo 21
Down syndrome (trisomy 21)
Turner syndrome (X0)
81
DNA testing + 3 marker types
tests typically involve examining genetic markers linked to disease-causing gene
- variable number tandem repeats
- restriction fragment length polymorphism
- single nucleotide polymorphisms
see that this version of a marker makes someone more likely to have a disease
82
Huntington's Disease
age it appears
symptoms
| autosomal _____
## Footnote
and starts with
autosomal dominant
genetically inherited neurodegenerative condition
Symptoms
- appear 30-50 years of age
- starts with subtle problems with mood and mental abilities
- rapidly advances to inability to talk, dementia, depression, and immobility
83
Testing for Huntington's
HD gene has a variable number of CAG triplets
>34 repeats = protein product with abnormal function = HD
- can be diagnosed by amplifying repeat region via PCR and analyzing the site of DNA amplicon on an agarose gel
84
Ethical considerations
HD is a good example of genetic testing being a double-edged
sword
* If your parent developed HD, would you prefer to know if you inherited the condition?
* If you found out you are a carrier and you are expecting a child, would you get your child tested before they were born?
* How would you react if you found out your child is a carrier? Would you treat that child differently?
* Some things may be better left unknown depending on your personality type
85
Eugenics
The idea of “enhancing humanity” by either encouraging select people to have children, or discouraging select people against having children
86
Genetic testing cons
Eugenics, genetic screening, and dystopian societies
Increased genetic screening, and genetic engineering methods may promote eugenics
If genetic data was more accessible to government or corporate agencies...
* Could healthcare be denied based on genetic data?
* Could insurance premiums increase for
those at risk of disease?
* Could employment be denied to those at risk of disease?
87
Genetic testing pros
* Newborn screening offers identification of rare diseases that can be treated and allow diagnosed children to live normal lives
* Knowledge from genetic screens may empower people to improve their lifestyle to minimize risks of disease and live healthier lives
* Genetic tests can reconnect long-lost relatives
* Increased knowledge and innovations in genomics could lead to
better cancer therapies
