Final Flashcards
1
Q
What are 3 things to consider when deciding on a model organism to use for a particular study?
A
Brood size, generation time, cost of maintenance
2
Q
Name 3 things that make mice a good model for human disease?
A
Inexpensive, small and easy to handle, similar genetics to humans
3
Q
Mice live ___ years and reach sexual maturity and _____ of age.
A
1-3 years
4 weeks
4
Q
Female mice are sexually receptive (or _____) every _____.
A
Estrous
4-5 days
5
Q
Mouse litters can contain _____ pups.
A
4-10
6
Q
Mice contain ___ autosomes.
Drosophila contain ___ autosomes.
Humans contain ____ autosomes.
A
19
3
23
7
Q
Mice chromosomes are _____ with the exception of which chromosome? Human chromosomes tend to be ______.
A
Acrocentric. Y chromosome. Metacentric
8
Q
Define synteny.
Define shared synteny.
A
Physical co-localization of genetic loci.
Conserved genetic context (arrangement of genes and regulatory elements) of particular loci between 2 organisms.
9
Q
__% of mouse and human genome can be aligned to regions of shared synteny. Approximately how many human genes have mouse homologues? Approximately what percent of human genes have drosophila homologues?
A
Over 90%
17,100 (70%)
75%
10
Q
What are 3 major advantages to using inbred mouse strains?
A
- Yields homogeneous (isogenic) genetic background
- Inbred mice aren’t sterile like other mammals tend to be
- They contain reduced phenotypic variation (valuable for studying disease causation)
11
Q
Male mice are mated with many females to ________.
A
Maximize the number of progeny. Also males produce more germ cells, so you increase the chances that a sperm will contain your transgene or modification of interest.
12
Q
The parental generation is called ____ in:
Mice?
Drosophila?
A
P0
P1
13
Q
What is the purpose of backcrossing?
A
To eliminate other variants and ensure that phenotype is due to the introduced genetic change, and nothing else.
14
Q
List 4 scenarios where backcrossing may be needed?
A
- If F1 is a chimera
- If P0 are from different strains
- If the allele is generated by random mutagenesis
- To eliminate off-target effects
15
Q
How many generations of backcrossing are needed to make a true congenic strain?
A
10
16
Q
What does it mean to be congenic?
A
An inbred strain of mouse that contains a small genetic region (ideally a single gene) from another strain, but which is otherwise identical to the original inbred strain
17
Q
Define transgene.
A
Genetic material transferred naturally or through a genetic engineering technique from one organism to another.
18
Q
What are the basic components of a mouse transgene?
A
- Promoter
- Intron
- 5’ UTR
- Gene of interest lacking introns (cDNA)
- 3’UTR/PolyA
- Enhancer
19
Q
Why include an intron in a transgene?
A
Has been shown to significantly increase the expression of the transgene and possibly the efficiency of RNA splicing
20
Q
What is required to target a transgene to a specific location within the genome?
A
5’ and 3’ homology arms
21
Q
What is the most commonly used method of random mutagenesis in mice?
A
Pronuclear injection
22
Q
What are the 6 steps involved in pronuclear injection?
A
- Fertilized mouse eggs are collected at the single cell stage
- Linearized transgene is injected into either male or female pronucleus
- Homologous recombination between transgene and mouse genome occurs
- Injected egg is transferred to a pseudo pregnant mouse
- Transgeneic (founder) mice are born that have transgene integrated randomly into their genome
- Genotype founders
23
Q
What are the steps involved in blastocyst injection of ESCs?
A
- ESCs are derived from the inner cell mass of a blastocyst
- ESCs are grown in 2D cell culture
- ESCs are transfected with transgene that contains a positive selection marker
- ESCs undergo positive selection, clonal expansion, and screening methods to detect the clones that contain the desired site-specific recombination
- The successful transgenic ESC clones are microinjected into a donor mouse blastocyst
- Injected blastocysts are transferred to a pseudo pregnant mouse
- Transgenic (chimera) mice are born that contain both injected ESCs and donor mouse cells
- Breed chimeras to WT parental mouse strain to get offspring that are heterozygous for the transgene
- Backcross heterozygotes to parental strain
24
Q
Pronuclear injection is a method used to study _____ muations while blastocyst inection of ESCs is used to study _____ mutations.
A
Gain of function
Loss of function
25
What is the difference between founder and chimera mice?
Founders generated through pronuclear injection and contain transgene in all cells
Chimeras are generated through blastocyst injection of ESCs and contain a mix of cells with transgene and from donor.
26
What are the pros and cons of blastocyst injection of ESCs versus CRISPR/Cas9?
Blastocyst injection Pro: Fewer off target effects
Blastocyst injection Con: Backcrossing is necessary, and this method is more costly/time ineffective, mutation might not allow for mouse development, the ES cells may not contribute to sperm development, mES lines are prone to chomosome loss/gain/robertsonian translocations
CRISPR Pro: No backcrossing necessary, new mutations can be easily added to existing mutant strains, and this is more cost/time effective.
CRISPR Con: More off-target effects
27
What is the ROSA26 locus?
A targetable locus in mice used to create a knock-in that allows for constitutive and ubiquitous expression of a transgene from intron 1 of this locus
28
Define reporter gene. What can they be inserted?
A gene that enables the detection or measurement of gene expression.
Downstream of promoter to measure promoter activity (but this may knockout endogenous gene)
OR
Downstream of gene, under its own IRES to measure both gene and promoter
OR
Fused to endogenous gene to measure localization of protein
29
What is the significance of using reporter mice?
Allows for the study of the transcriptional activity of a promoter or for the monitoring of protein localization or trafficking in vivo.
30
What is the difference between a knock down and a knock out?
Knock down: gene expression is reduced at the RNA or protein level (RNAi)
Knockout: gene expression eliminated at the nucleotide level (CRISPR)
31
LoxP sites are __ bp sequences that consist of _____bp____ sequences that flank a ___bp__ sequence.
34
two 13-bp inverse repeat
8-bp core
32
If LoxP sites are in the same direction, what happens to the sequence in the middle?
It is excised.
33
What are the advantages (@) and disadvantages (7) of CreLoxP
Advantages: Works in almost any cell type, can be used in conjunction with other systems (i.e. tet inducible Cre), contains only 2 major components.
Disadvantages: Off target tissue activity, leaky expression, irreversible, potential phenotypes due to cre expression alone, numerous steps necessary for construct and mouse strain construction, can stop working/will silence over generations, recombination efficiency of LoxP sites can be affected by postition and distance between 2 sites leading to mosaicism
34
What components are necessary for Tet on/off systems?
1. Tetracycline responsive element (TRE) that is 7 repeats of 19bp tet operator sequences
2. Tetracycline transactivator/reverse transactivator
3. Tetracycline or a tet derivative (doxycycline)
35
What happens when tetracycline is added in a tetOFF system? How about a tetON system?
TetOFF: gene is not expressed in presence of tet, since tet binds to tTA
TetON: gene is expressed in presence of tet since tet binds to rtTA and allows it to bind and activate gene expression
36
What are the advantages and disadvantages of TetOn/Off systems?
Advantages: Reversible, gives you more temporal control than CreLoxP
Disadvantages: Can yield leaky expression, tet can be toxic at high levels, or administration at the wrong time during development.
37
What are the advantages and disadvantages of auxin inducible degron systems?
Advantages: reversible, fast
Disadvantages: Not in use for mouse models, you need to create an AUD protein
38
What components are necessary for the auxin inducible degron system?
1. Ter1 (transport inhibitor response 1) - promotes interaction between E3 ubiquitin ligase and auxin
2. E3 ligase
3. Auxin - a plant hormone that can mediate the interaction of Ter1 and the auxin inducible degron
4. An auxin inducible degron tagged to protein you want to degrade.
39
CreLoxP works on ____,
TetOn/Off works on ____,
Auxin inducible degron systems work on ___.
DNA
RNA
Protein
40
What is ENU and how does it act as a mutagen?
N-ethyl-N-nitrosourea is an alkylating agent that transfers its ethyl group to one of a number of reactive sites on DNA nucleotides, including the O6 of guanine to produce O6-ethylGuanine (O-eG). This causes a CG to AT transition that is passed on after spermatogenesis.
41
What are E-males?
C57BL/6 male mice that have been treated with ENU.
42
What happens after E-male exposure to ENU?
They recover their fertility for 12 weeks before being mated to C57BL/6 or C3H females.
43
What is the general mating scheme for ENU mutagenesis?
E-male mated to C57BL/6 or C3H female.
G1s are heterozygous for unique ENU-induced mutations. If the mutation is dominant, it will show up in G1.
If you're looking for a recessive mutation, mate G1 male with a C57BL/6 or C3H female to yield heterozygoys and WT G2. Cross female G2s with a G1 male before screening.
44
If looking for a recessive mutation during ENU mutagenesis, what is the frequency of a given mutation at the G3 stage? How many G3s will be necessary to ensure that a mutant is identified?
1 in 8.
16-36.
45
In what scenario would you cross the C57BL/6 E-male with a C3H female instead of a C57BL/6 female?
If you want to introduce variation early or if you want to map mutations.
46
How does gene mapping woth C3H mice work?
Outcross a G3 mouse with a C57BL/6 background that is homozygous for your mutation with a C3H mouse to yield heterozygous G4 mice. Cross these with the affected G3 animals (or intercross the hets) to identify G5 mice with and without the phenotype of interest and genotype these mice using STRs or SNPs which allows determination of linkage of the mutation with a particular marker.
Or, if the G3s were already crossed to C3H previously, mapping data can be obtained directly from G3 mice.
47
ENU mutagenesis produces ____ mutations which allows for the discovery of what types of mutant alleles?
Point mutations.
Hypomorphic or separation of function (novel) alleles.
48
ENU mutagenesis is particularily useful for generating ____ alleles of genes involved in ______.
Hypomorphic
Development
49
How do you generate a pseudopregnant mouse?
Mate her to a vasectomized male.
50
What is the fastest method of creating transgenic mice? How does this work?
Testis electroporation
Linearized DNA is injected directly into testis and electroporation (pulses of high voltage electric shocks) introduces the DNA into germ cells. Progeny can be screened via GFP and PCR genotyping.
51
What are the advantages (6) and disadvantages (4) of transgene integration?
Advantages: fast, easy, high-throughput, can introduce foreign genes, can screen for dominant negative alleles, ad can test promoters for expression.
Disadvantages: random integration, multiple integrations, disruption of critical regions of the genome, and silencing of transgene due to region of integration.
52
What are the 3 main features of a targetting vector?
5' homology arm and 3' homology arms flanking a positive selection gene (i.e. NEOr), all upstream of a negative selection gene (i.e. HSV-ik)
53
What is the purpose of negative selection in gene targetting of ESCs? Positive?
To select against cells that have integrated the transgene randomly.
To select for cells that were transformed.
54
What steps you would take to verify mESCs cells prior to injecting mESCs into a recipient blastocyst (6).
1. Select for Neo resistance and absence of HSV-tk expression
2. Karyotyping to look for structural abnormalities
3. PCR verification of 5’ and 3’ arms
4. Sequence – Whole genome, relevant region
5. Southern Blot (use Neo as radioactive probe)
6. Look at cellular morphology – if abnormal, they have differentiated
55
p53 mutants are susceptible to _______.
Spontaneous tumors, especially upon carcinogen treatment
56
What's the major disadvantage of a knockout mouse? How can this be somewhat fixed (give 2)?
Loss of gene function can often result in lethality.
If a heterzygote results in haploinsufficiency OR create a conditional knockout allele.
57
What components are required for a FRT/Cre-LoxP system?
1. Cre (under tissue/cell specific promoter)
2. LoxP flanking gene of interest
3. 5' and 3' homology arms
4. Neo resistance cassette
5. LacZ reporter
6. FRT (flp recombinase sites) flanking LacZ
7. FLP
58
What is the purpose of the knockout first/knokout ready alleles?
Knockout first alleles (tm1a) have the gene of interest non-functional since LacZ and Neo are blocking expression. Once flp recombinase is added, LacZ and Neo are removed to generate a knockout ready allele (tm1c) that is flanked by lox P sites, so that a particular exon or part of the gene of interest can be removed with Cre recombinase.
59
Cre that is inserted downstream of Cfd9 will be expressed in what tissue/cell type?
In oocytes during maturation
60
ERt2-Cre transgenes is a fusion between cre recombinase and _____. This resulting protein is confined to the ____ (cellular location). In the presence of _____, what happens to CreER?
Estrogen receptor.
Cytoplasm.
Tamoxafin
Translocated to nucleus and can act on LoxP sites
61
What things are needed to generate CRISPR-Cas9 mice?
1. Cas9 protein
2. crRNA + tracrRNA (gRNA)
3. ssODN (oligodeoxynucleotides) repair template/ insertion template.
62
What is a coisogenic strain?
What is a congenic strain?
What is a consomic strain?
What is an inbread strain?
A strain that differs from an established inbred strain by a mutation at only one locus
A strain formed by backcrossing to an inbred parental strain for 10 or more generations while maintaining heterozygosity at a selected locus.
A strain in which one chromosome has been replaced with the homologous chromosome from another strain.
A strain that has been maintained by sibling (sister x brother) matings for 20 or more consecutive generation
63
How do you superovulate a mouse?
Inject mouse with equine chorionic gonadotropin (eCG), then inject with human chorionic gonadotropin (hCG) and harvest after 12-14 hours
64
What are SMC protein complexes?
```
Structural Maintenance of Chromsomes (SMC) - cohesion is an example of one - that functions in:
DNA replication
DNA damage repair
Chromosome condensation
Chromosome segregation
Transcription
```
65
What are SMCopathies?
```
Affects ~ 1 in 10,000 births
Defined by:
Microcephaly
Cognitive retardation
Growth retardation
Organ defects
Cancer predisposition
```
66
What does a gene trap allele contain?
1. A promoterless reporter gene and/or selectable genetic marker (beta-geo)
2. An upstream 3' splice site acceptor
3. A downstream transcriptional termination sequence (polyA).
4. A negative selection marker (HSVik)
67
What is the function of a gene trap, and list 2 ways in which this is useful.
GT is inserted into an intron of a GOI and the cassette is transcribed from the endogenous promoter of that gene in the form of a fusion transcript in which the exon(s) upstream of the insertion site is spliced in frame to the reporter/selectable marker gene. Transcription is terminated prematurely at the inserted polyadenylation site so that the fusion transcript encodes a truncated version of the cellular protein and the reporter/selectable marker.
Gene traps simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a DNA tag for the rapid identification of the disrupted gene.
68
What is Beta-geo? What is its major disadvantage? How can this be mitigated?
β-galactosidase and Neomycin resistance fusion protein
This is a selection, so expression of b-geo is dependent on expression of protein X (which makes it difficult to select)
Tag Neo (the selection part) to its own constitutive promoter.
69
What are 6 advantages to using drosophila as model organisms?
1. Small - easily maintained and handled
2. Short life cycle
3. Numerous offspring
4. Cheap
5. Simple genome
6. few legal and ethical restrictions
70
A normal fruit fly lives _____ days. Their life cycle is _____ dependent.
40-50 days
| Temperature
71
How many days does it take a fertilized embryo to become a normal fly at 25 degrees? How about at 18 degrees?
10
Twice as long
72
What are the stages of fly development? How long does it take to get from one stage to the next?
```
Egg/embryo (Day 1)
1st instar larva (Day1-Day 2)
2nd instar larva (Day2-Day 3)
3rd instar larva (Day3-Day 5)
Pupa (Day 5-Day 10)
Adult
```
73
When will flies begin mating?
10 hours after eclosion
74
Why are virgin female flies used?
Females can store sperm from past matings so it is important that this is not a confounding possibility.
75
What characteristics allow for visual distinction between male and female and virgin female fruit flies?
Females: tend to be larger, striped and pointed abdomen.
Virgin females: Abdomens are whiter and larger and contain a black spot called a meconium.
Males: TBlack and rounded abdomen, have sex combs on legs
76
How does the Y chromosome act differently in fruit flies than humans?
Y doesn't determine maleness in flies.
77
What are polytene chromosomes? Where are they found?
Generated by multiple rounds of DNA replication without cell division in the cells of salivary glands
78
List 4 functions of a balancer chromosome.
1. Keep lethal or sterile mutations in heterozygous
2. Avoid meiotic recombination by containing multiple inversions
3. Allow for visually following genotypes in a cross since they contain dominant markers
4. Are homozygous lethal in adult flies, but are viable as embryos, so useful for studying development.
79
In balancer notation, what do F, S or T stand for?
First
Second
Third (chromosome)
80
Balancer chromosomes are homozygous lethal. How can you maintain them?
Keep them in trans to a different dominant marker on the same chromosome
81
How do balancers mitigate recombination?
Contain inversions so that if recombo occurs, an acentric and dicentric chromosome is formed and the offspring is inviable.
82
How can mapping be done in flies?
1. Complementation/Deletion mapping
2. Recombination/linkage
3. Sequencing
83
Complementation mapping uses ____ in what organism? What can it also uncover?
Plasmids. Yeast.
Suppressors.
84
Plasmids are transformed at a ____ratio of plasmid to cells (approximately ___ plasmid/cell)
Low
1
85
In recombination mapping _____ can be used as a proxy for relative distance.
Recombination frequency
86
What sex should you pay special attention to in recombination studies in flies? Why?
Only female flies can undergo recombination.
87
How do you calculate recombination frequency for recombination mapping? What units are used?
Recombination frequency = # of recombinants / total # flies = map units or cM *** multiply double crossovers by 2***
88
The most common offspring in a recombination mapping experiment will be recombinants or non recombinants?
Non recombinants
89
How can you improve the estimation of distance in recombination mapping? (2)
1. Many offspring
| 2. Many recessive markers across the genome
90
After mapping, how can you confirm your candidate gene? (4)
1. show that gene variant segregates with phenotype
2. Sequence many mutants with a WT and determine if all mutants have a mutation on the same gene
3. Rescue a mutant with a candidate gene (a suppressor would do this too) (drosophila use UAS-Gal4)
4. Does a knockout of the candidate gene recapitulate the mutant phenotype?
91
What are the cons of the 3 different types of gene mapping?
Complementation Con: Requires plasmid libraries available
Recombo Con: many offspring required
Sequencing Con: Expensive
92
Explain the concept of complementation mapping, recombination mapping, and sequencing mapping
Complementation: rescue or fail to rescue mutant phenotype by providing an exogenous source
Recombination mapping: measure distance from marker genes by observing meiotic reombo
Sequencing: sequence several specimens to find a gene where each specimen has a variant.
93
Define break point in deficiency mapping.
Where the deficiency starts and ends
94
Deficiency lines that are homozygous are often _____. How can you overcome this and be able to distinguish which flies have the deficiency and which ones don't?
Lethal
Use balancer chromosomes.
95
How many genes are removed in a deficiency?
dozens to hundreds
96
Why are there balancer stocks for 90% of the genome but not 100%?
Some genes will be lethal as haploinsufficient/heterozygotes.
97
How can deficiencies be used for mutation mapping?
1. EMS mutagenize
2. Screen for desired phenotype
3. Determine complementation groups
4. Mate deficiency:GFP to EMS:GFP
5. Pick non GFP and see if the embryo is normal (EMS and Df complement) or abnormal (EMS and Df fail to complement and mutation is within the deficiency region)
98
The UAS-Gal4 system is endogenous to which organism? This gives it specificity in which organism?
Yeast.
Fly
99
UAS is a ___ ____ which is a _____ _____ sequence.
GAL4 enhancer
Regulatory DNA
100
GAL4 is a ____ ____ that is encoded elsewhere in the genome.
transcription factor
101
What are 5 main things that UAS GAL4 can help study?
1. Overexpression of a specific gene
2. Can study gene expression in specific tissues
3. Can be used to rescue gene knockouts (or find suppressors)
4. Can be used to express RNAi for tissue specific knockdowns
5. Can use GAL80 for temporal control
102
What is the difference between necessary vs sufficient?
Necessary = deleting gene creates mutant phenotype
Sufficient = rescuing mutant by introducing your gene of interest
103
What is a P element?
A class II (cut and paste) transposable piece of DNA found in flies similar to human Alu.
104
What are the components of a P element?
1. ORF coding for transposase
| 2. 2 inverted repeats flanking the ORF
105
Where do P elements insert? What types of mutations can they cause?
At consensus sites near TSSs
1. Mutations during insertion (disrupt insertion site)
2. Mutations due to imprecise excision (NHEJ)
106
What are 3 main things that P elements can help study?
1. Create mutations (insertion/excision) by replacing transposase with GOI
2. Provide a WT copy of a gene or other DNA
3. Report on gene transcription (enhancer trap)
107
Why are they called "P" elements?
Found from males (paternal) from a newly isolated strain from the wild
108
If you want to excise a P element containing the "white" gene, what type of fly should you cross this with?
One that has transposase linked to a dominant marker. Then cross these offspring to a fly with balancer chromosomes and select flies with white eyes (P element is gone) and lacking the transposase marker.
109
What are enhancer traps?
P elements that have inserted between an enhancer and a gene, so that the enhancer acts on the P element and expresses the GOI (usually GFP or Gal4) in that P element so that the expression patterns of enhancers can be studied.
110
What can you do to map enhancer traps/where P element was inserted? (4)
1. Isolate genomic DNA
2. Digest genomic DNA
3. Inverse PCR performed with primers inside the GFP-p-element (that may be tagged with antibiotic resistance to select only for plasmids with the p element)
4. Sequence and map
111
# Define clonal analysis
What organisms can it be used in?
A technique used to create a mosaic organism in which some cells are mutant for a gene of interest amongst other cells that are WT
Flies, zebrafish, mice
112
What are 5 main things that clonal analysis can help study?
1. Function of essential genes in adults
2. Which cell types are the causative cells
3. Determine if a mutation is cell autonomous or non autonomous
4. Cell competition (i.e. cancer and tumor suppressor genes)
113
What does it mean when a mutation is cell autonomous vs non-autonomous?
Autonomous - mutation in a cell type causes a phenotypic change in that cell type
Non-autonomous - mutation in a cell type causes a phenotypic change in another cell type (usually in ligands and pathways)
114
What technique can be used to increase recombination frequency?
FLP-FRT
115
What is MARCM? What is this used for and what other things can be used to do this?
Mosaic analysis with repressible cell marker. Uses a marker to positively mark clones through the loss of a repressor.
To identify mutant clones. Antibody to protein of gene, marker to create negatively marked clones
116
How many eggs can a fly lay in a day?
100
117
What 3 things are highly conserved between flies and mammals?
Tissues, development, and brain functions
118
What is fly chromosome 4?
A dot chromosomes that is mostly heterochromatin and carries only 1% of genes
119
Which 2 fly chromosomes are mostly heterochromatin?
4 and Y
120
List the number ranges associated with each fly chromosome arm.
```
X = 0 - 20
2L = 21 - 40
2R = 41 - 60
3L = 61 - 80
3R = 81 - 100
```
121
In flies, recessive mutations are written ____ while dominant mutations are written ____. Allele names are _____.
Lowercase.
Uppercase.
Superscripted.
122
How can mutations be generated in flies? (4)
1. EMS
2. X-rays and gamma rays
3. Transposons
4. Homo recombo and CRISPR/Cas9
123
List pros and cons of:
1. EMS
2. X-rays and gamma rays
3. Transposons
4. Homo recombo and CRISPR/Cas9
1. Pros= random, saturation, different types of mutants.
Cons = slow gene ID, large scale
2. Pros = random, gene dels and dups.
Cons = slow gene ID, no real saturation, inefficient
3. Pros = fast gene ID, flexible scale
Cons = non-random (hot spots), no saturation
4. Pros = CRISPR is highly efficient
Cons = currently inefficient for screening :(
124
What sex of flies are most useful for EMS screens?
Males, each sperm will have a different mutation
125
Why should you never keep +/Balancer stocks?
You will lose the balancer eventually - need 2 balancers together or a balancer and a mutation.
126
Transposase mRNA is only spliced in _____ cells but not ____ cells.
Germ.
Somatic.
127
Recombination occurs in ____ of the cell cycle. It can be enhanced by ____ and ____.
G2
FLP-FRT and X rays
128
Which methods can be used most effectively as reverse genetic techniques in flies?
RNAi - hypomorph (lof)
| CRISPR - null
129
Mendelian disorders are caused by a ____ locus and follows Mendel's laws...which are?
Single.
1. Law of Dominance
2. Law of Segregation
3. Law of Independent assortment
130
List 6 factors that can complicate pedigree interpretation.
1. Incomplete penetrance
2. De novo mutations
3. Adult-onset conditions
4. Germline mosaicism
5. Anticipation
6. Pleiotropy (gene has multiple effects)
131
Which inheritance patterns are mendelian and which ones are non-mendelian?
Mendelian: Autosomal dominant, autosomal recessive, X-linked dominant, X-linked recessive, Y-linked
Non-mendelian: mitochondrial, multifactorial, imprinting
132
What occurs during imprinting?
Paternal: paternal allele is silenced and mother is the carrier for the disease allele
Maternal: maternal allele is silenced and the father is the carrier for the disease allele.
133
How can heritability be calculated? What are the variables that need to be determined?
VG / VP = heritability^2
```
VG = genetic variance
VE = environmental variance
VP = phenotypic variance (VP = VG + VE + 2Cov(G,E))
```
134
What is the difference between linkage and association for mapping studies in humans?
Linkage: Follows meiotic events (recombination) through families to test for co-segregation between marker alleles and disease phenotype. Most powerful when studying a highly penetrant phenotype in large families
Association: tests for differences in frequency of genetic markers in groups of unrelated individuals (population). Typically used to find common, low penetrance alleles that increase disease susceptibility
135
The current human reference genome is (diploid/haploid) and was assembled using _____.
Haploid.
Shotgun sequencing.
136
Indels are ____ bp in size
| CNVs are ____bp in size
less than 50
more than 1000 (or 1kbp)
137
Chromosome abnormalities can fall into which 2 classes?
Numerical (aneuploidies)
Structural
138
What is hardy weinberg equilibrium?
p2 + 2pq + q2 = 1
139
List the assumptions of HWE (5)
1. No selection (no differences in chance of survival)
2. No mutation (no new alleles introduced)
3. No migration (no new alleles introduced)
4. Infinite population (genetic drift is not causing random changes in allele frequencies)
5. Random mating (part of the "no selection" thing)
140
How can you calculate allele frequency from genotype counts?
Frequency of A = frequency of AA + (frequency of Aa/2)
| Frequency of a = frequency of aa + (frequency of Aa/2)
141
Why might populations deviate from HWE? (4)
1. Inbreeding
2. Genetic drift (small populations or alleles with low allele frequencies aren't passed on)
3. Population fragmentation/founder effects (populations are split)
4. Mutation
142
How do you calculate the degrees of freedom for HWE ? What is the df for a biallelic locus?
df = # of genotypes - # of alleles
1
143
How do you calculate the chi2 for HWE?
(obs-exp)^2 / exp
144
What is it called when more than one population is involved in HW?
Population substructre
145
When is it important to identify multiple HW populations in a cohort?
Important for genetic association studies
146
What results from a chi square test would indicate a significant deviation from HWE?
A very high number
147
If a population has a much higher incidence of homozygotes of the minor allele than expected, what is a possible explanation?
More than 1 population being studied (population substructure)
148
Give 2 reasons why alleles can differ in frequency between groups
Founder effects and genetic drift
149
Give 2 reasons why false positive disease associations can be made from ancestry
Different rate of disease between ancestry groups and chance oversampling of one population in ascertaining cases or controls
150
What does HWE describe?
The theoretical relationship between genotype and allele frequency in a single, large population
151
The human reference genome is ____ bp.
3.3 billion
152
What makes genome assmbly difficult in humans?
Repetitive sequences
153
What makes a SNV a SNP?
A MAF of > 1%
154
Define haplotype
A set of alleles that tend to be inherited together (rarely recombine) in one contiguous DNA sequence.
155
Define genetic drift
Changes in allele frequency based on random differences in allele passage from one generation to the next.
156
What is Fst? How do you calculate Fst?
Fixation index: divergence between populations
Fst = variance in allele frequency between subpopulations weighted by size / variance in allelic state across all individuals
157
What is the Fst for a population in HWE?
0
158
What 2 things can decrease fixation index in humans
Interbreeding between subpopulations (affected by geographic and cultural factors in humans), and the time since reproductive isolation
159
About ___% of genetic differences between any two people are the result of ______.
15%
Continental ancestry
160
What are 2 methods that can be used to determine individual ancestry?
PCA and Bayesian clustering
161
What is phasing? Can it be done without parental genotypes?
Placing an individual's alleles (which could be on either chromosome) onto the maternal or paternal chromosome.
yes, can use haplotypes from a large collection of ancestry-matched control samples
162
What does it mean when an allele is identical by state?
Identical alleles in two or more individuals
163
What does it mean when an allele is identical by descent?
Alleles shared between two individuals/genomes resulting from shared inheritance of those alleles from a common ancestor.
164
What is IBD0, IBD1, and IBD2?
Identical by descent at neither allele, 1 allele, or both alleles
165
What is the kinship coefficient? How is it calculated?
The probability that a randomly chosen allele from each of two individuals will be IBD
Kinship coefficient = sum (1/2)^n for each common ancestor where n is the number of individuals in the path from 1 individual to the other through their common ancestor
OR
= (1/4)IBD2 + (1/2)IBD1
166
What is the inbreeding coefficient of an individual?
The same as the kinship coefficient of her parents
167
How does the inbreeding coefficient change if a common ancestor is the product of a consanguineous union?
multiply the inbreeding coefficient by (1 + kinship of that individuals parents)
168
Some individuals that have the allele have the disease and others don't. What is this called? This is characteristic of what type of inheritance pattern?
Incomplete penetrance
Non-mendelian inheritance
169
What are the 2 major pitfalls of GWAS?
1. Population stratification (differences in alleles due to sampling of different populations
2. Multiple testing (if you test something many times, even fairly low-probability events will happen many times.
170
What is the result of strong positive selection?
A selective sweep where the advantageous allele is maintained, but so are flanking regions around the mutation.
171
What is the likely cause of CNVs? How can you assess CNV?
Aberrant recombination. Comparitive genomic hybridization
172
How are mono and dizygotic twins formed?
```
Mono = early fissioning of 1 egg
Di = occasional ovulation of 2 eggs
```
173
Regardless, mono and dizygotic twins have a chance of sharing ______. How is this beneficial?
Circulation
All blood cells in common - can use each other perfectly for transplants
174
How are hybrid chromosomes generated?
Non-homologous recombo in meiosis
175
What is the most appropriate way to assess disease causing mutations that:
1. Are rare and very damaging
2. Are rare and not damaging
3. Are common and not damaging
4. Are common and very damaging?
1. WES, WGS
2. We don't find them (no diesease)
3. GWAS
4. We don't find them (natural selection)
176
Rare variants make up __% of the human genome?
0.01
177
What types of diseases/mutations can't be studied using WES?
Aneuploidies
CNVs
Polygenic diseases resulting from large indels (doesn't work because before WES the DNA is sheared and WES generates relatively short reads)
MtDNA diseases (since only the nuclear genome is captured)
178
what types of proteins are particularly susceptible to dominant negative/antimorph mutations?
Multimeric proteins composed of 2 or more polypeptides joined together to form a functional protein
