Old exam 1 questions Flashcards
(115 cards)
1
Q
In E. coli DNA replication, the ring-shaped hexameric DnaB helicase travels along the single-stranded DNA in the 5’ to 3’ direction and unwinds double-stranded DNA at the replication fork as it migrates. In a standard replication fork, the strand of DNA that DnaB travels on serves as a template for the synthesis of what nascent molecule?
A- The leading strand of DNA
B- The lagging strand of DNA
C- Either strand of DNA
D- A strand of mRNA
E- There is not enough information
A
B the lagging strand
2
Q
Draw a cartoon schematic of the replication fork with DnaB at work to illustrate your point (that DnaB is traveling on the template strand for lagging strand), marking the directionality of the parental and nascent DNA strands.
A
5’ —————-O———3’
3’ <–<–<–<– |
5’ ————–>|
3’ —————————5’
O = DNA B, on the template strand for synthesis of the lagging strand
3
Q
Why do you think the prime editing system is less constrained by PAM than conventional CRISPR/Cas gene editing systems?
A
In conventional CRISPR editing, the position of the edit is dictated by the cut site (which takes place 3-4 nt upstream of PAM in the Streptococcus pyogenes system). In prime editing, the position of the edit with respect to PAM is dictated by the RT template of pegRNA (that gets copied by the RT (fused to Cas9 nickase) starting at the nicked site and extending for the entire length of the RT template). pegRNA can harbor a fairly long RT template (30nt+), with an edit in the nicked DNA introduced anywhere between the nick site and the 5’ end of the RT template (i.e. immediately after the nick site, in the PAM itself or significantly downstream of PAM, as far as the RT template allows).
4
Q
Why do you think the human telomerase exists as a dimer, with two TERC and two TERT subunits present?
A
The two TERC/TERT units of telomerase work in parallel on the ends of two sister chromatids to ensure equal telomere extension.
5
Q
In the 1958 Messelson-Stahl DNA replication experiment discussed in class (where E. coli grown in heavy 15N was moved to a light 14N source and a shift in the density gradient centrifugation pattern of genomic DNA was examined over time), what percentages of light, intermediate and/or heavy DNA do you expect to see after 2 hours (6 generations) of cell division in 14N? Assume that E. coli divides every 20 min.
A
0% heavy, 3% intermediate, 97% light
6
Q
List four molecular technologies discussed in class that rely on emulsion PCR.
A
454 pyrosequencing
SOLiD sequencing by ligation
Ion Torrent sequencing by synthesis
Digital droplet PCR (ddPCR)
7
Q
The copy number of DNA Pol III in E. coli is estimated at 10-20 copies of this multi-protein complex per cell. Why do you think the cell needs more copies of this essential protein complex than there are replication forks on its chromosome?
A
To participate in plasmid replication and in DNA repair.
8
Q
A generic name of an enzyme that fuses the 3’-OH end of one DNA molecule to the 5’-P of another DNA molecule
A
DNA ligase
9
Q
A generic name of an enzyme that removes the 5’-P on the molecule of DNA
A
phosphatase
10
Q
A hybridization-based molecular method covered in class to study RNA distribution with cellular resolution in a slice of tissue
A
RNA in-situ hybridization
11
Q
A fluorescence-based protein-protein interaction method that takes advantage of non-radiative energy transfer between two fluorescent proteins
A
FRET
12
Q
A bacterial enzyme involved in nick translation
A
DNA Pol I
13
Q
A eukaryotic enzyme that removes a stretch of single-stranded RNA/DNA displaced by DNA polymerase delta prior to Okazaki fragment joining
A
FEN1
14
Q
A next-gen sequencing method that detects protons being released during DNA synthesis
A
ion torrent
15
Q
Two kinases that phosphorylate eukaryotic replication licensing factors
A
S-phase CDK and Ddk
16
Q
A generic name of a marker gene that assists in the identification or propagation of plasmids in bacterial or fungal strains with specific nutrient requirements
A
auxotrophic marker
17
Q
A modern sequencing technology that enables the detection of specific chemical modifications in DNA or RNA
A
oxford nanopore or pacbio SMRT
18
Q
A hexameric protein complex in eukaryotes that marks the origin of replication throughout the cell cycle
A
ORC1-6
19
Q
A molecular method that determines the coordinates of a DNA-binding protein on its target DNA
A
DNase I footprinting (bp resolution level) or ChIP-seq/DAP-seq (maps binding to a 200-500bp region
20
Q
An outdated gene expression quantification method that can be used to map how far the ends of a transcript extend
A
RNase protection assay
21
Q
A protein in eukaryotes that confers processivity to replicative DNA polymerases delta or epsilon.
A
PCNA
(Proliferating cell nuclear antigen)
22
Q
A DNA secondary structure that forms in repetitive sequences harboring direct tandem repeats
A
Slipped structure
23
Q
A bacterial restriction enzyme utilized in two different DNA editing technologies
A
Fok1
24
Q
A medium/high-throughput gene expression analysis method covered in class that involves an affinity purification step
A
nanostrings
25
A ribonuclear protein complex in eukaryotes that builds telomeric ends
Telomerase
26
A chemical property of a DNA double helix that has the most contribution to its stability
Base stacking due to their hydrophobicity
27
Why do you think the oriC of one bacterial species may not work in another bacterial species? How will you experimentally test in vivo and/or in vitro that your explanation is mechanistically correct?
DNA elements within oriC could have diverged in evolution and may not be properly recognized by other species’ DnaA (and other factors like IHF). If that is the case, then protein-DNA interaction assays like EMSA between the A-box and DnaA from the same and divergent bacterial species will show a lack of binding with mismatched protein-DNA pair, but good binding with matching combinations. For these experiments, A-box-containing DNA fragments can be amplified and labelled by PCR, whereas the DnaA protein can be expressed from a recombinant DNA construct in a cell-free in vitro transcription-translation reaction (this is to avoid the possible toxicity of DnaA when expressed in bacteria).
An alternative explanation is that the spacing between the DNA elements is different in different species (e.g. due to a slightly different size and hence the footprint of DnaA) that ultimately prevents proper oligomerization between DnaA monomers. The footprint of DnaA on the oriC fragment can be examined by DNase I footprinting and the oligomerization can be tested by EMSA (a supershift is expected) or a biochemical technique that we have not discussed in class called gel filtration (that separates protein (or in this case, protein-DNA) complexes by size). Other protein-DNA binding techniques (e.g., yeast-1-hybrid) can be employed to survey DnaA/A-box binding compatibilities of matched and mismatched DNA-protein combinations. PCR-based site-directed mutagenesis (a technique that introduces desired mutations via the primer sequences) can be performed to change DnaA sequences or the spacing between the A-box elements to see the effect of these mutations in vitro or in yeast on DnaA binding from both bacterial sources.
Note that working with oriC mutants in the native chromosomal context is probably not realistic, as the lack of chromosome replication (that may result from sequence edits) means the bacterial cultures will fail to grow (and there won’t be any DNA to even confirm that the edits took place let alone study replication dynamics). If you want to study oriC mutants in bacteria, you can use an oriC-containing plasmid instead and select for the plasmid using an antibiotic resistance marker gene.
28
Screening for protein-protein interactions using yeast 2 hybrid is often problematic when using transcription factors of interest as baits. This is because transcription factors can transactivate the reporter even in the absence of any bait-pray interaction. Can you suggest two experimental workarounds that bypass this transactivation issue and enable you to test for interactions between a transcription factor of interest and its possible partner using yeast 2 hybrid?
1. Swap the bait and the prey. Placing a transactivating transcription factor in the prey vector and the putative interactor in the bait vector solves the problem.
2. Subdivide the transcription factor into individual overlapping domains and use those domains that do not transactivate as baits to screen against the prey.
3. Fuse the prey to a strong repressor (rather than an activator) domain and screen for the shutdown of the reporter activity upon the successful bait-prey interaction (the repressor domain of the prey with override the transactivation ability of the interacting bait) using colorimetric and fluorescent markers or a URA3 gene in combination with a chemical called 5-FOA. 5-FOA is toxic to cells with active URA3, so the yeast cells will only survive if they repress URA3 activity upon bait-prey binding.
4. Use a transcriptional activation system that is based on RNA polymerase III (that normally transcribes tRNAs and other housekeeping non-coding RNAs) rather than RNA polymerase II (that transcribes protein-coding genes). The assumption here is that the bait transcription factor of interest that transactivates RNA pol II promoters should not transactivate an RNA pol III promoter, so switching to a Pol III system solves the transactivation problem. The pol III system relies on a prey construct fused to a subunit of a general transcription factor TFIIIC that indirectly (via TFIIIB) recruits RNA Pol III and turns the transcription of a reporter gene on if recruited to the DNA via the prey-DBD interaction. The reporter is driven by the regulatory promoter elements recognized by TFIIIC and TFIIIB, enabling the activation of this system upon bait-DBD and prey-TFIIIC interaction.
29
Imagine that you just joined a new lab that studies flower development and your first task is to determine if the gene PRETTY1 is expressed in flower samples from 10 rare (not sequenced) tropical species. Assume that PRETTY1 homologs have been previously sequenced and characterized in several model organisms and shown to be important for petal shape. You do not know the exact sequence of PRETTY1 in any of these rare tropical species (and have no budget to sequence them), but you do have multiple sequences (and clones) of PRETTY1 from several model plant species and know that the sequences of PRETTY1 vary between different plants but are all related. How will you go about testing whether PRETTY1 homologs are expressed in the flowers of valuable tropical samples? Propose two experimental approaches you can use and clearly state any protocol modifications that would be required to detect PRETTY1 being expressed (Hint: mentally transpose yourself in time to the past and think how scientists used to approach these types of questions before next-gen sequencing was invented).
cDNA sequences from all available PRETTY1 gene homologs from different species can be computationally aligned to one another and the most conserved regions of the sequences determined. The conserved regions can be used for developing a hybridization probe for a Northern blot and for designing a pair of degenerate primers* that would recognize the PRETTY1 gene from all or most species (*degenerate primers are pools of primers with every possible nucleotide present in each non-conserved position of the primer sequence).
Northern blot: Because all PRETTY1 sequences are related, a labelled DNA probe from a model species’ PRETTY1 (amplified from model organism’s RNA by RT-PCR or from a cDNA clone by PCR in the presence of labeled nucleotides) can be hybridized to a Northern blot where RNAs from 10 tropical flower samples (and a control model species RNA) were resolved. By using a lower temperature and higher salt during the probe hybridization and washing steps (less stringent conditions), binding of a partially complementary probe will be enabled and can be detected by autoradiography.
RT-PCR: Reverse transcribe RNA of the 10 species of interest (plus from a model organism as a positive control) using oligo-dT and then employ degenerate primers against the most conserved regions of the PRETTY1 gene for the amplification of cDNA fragments from all 11 RNA samples. The RT-PCR products from each species can be Sanger sequenced, new internal (aka nested) gene-specific primers designed and used for gene-specific RT-qPCR on the 10 samples (and a positive control) to evaluate PRETTY1 expression across all tropical genotypes.
In-situ hybridization: Using gene-specific PRETTY1 primers developed for the RT-PCR approach above, amplify short cDNA fragments of each tropical PRETTY1 gene (and a model plant control) into a bacterial vector that harbors specific RNA polymerase promoters (e.g., T3 and T7) surrounding the cloning site (subcloning is not a requirement, as T3 and T7 promoter sequences can be introduced at the flanks of the linear PCR fragments via PCR). Generate DIG-labeled sense and antisense RNA probes by in vitro-transcribing the cDNA fragments using T3 and T7 RNA polymerases. Hybridize the probes (sense and antisense probes for 11 PRETTY1 genes, including the positive control) to the slices of embedded, permeabilized flowers for the 11 respective species and detect the PRETTY1 signal in 22 in situ hybridization reactions using commercial anti-DIG antibodies coupled with an enzyme (e.g. horseradish peroxidase). Run an enzymatic assay and detect the PRETTY1 signal by monitoring the enzyme activity. The sense PRETTY1 probe is used as a negative control for the antisense probe complementary to the PRETTY1 transcript of interest.
30
Imagine that your lab is characterizing a new immortal cell line derived from a dog melanoma. You are tasked with examining the length of the telomeres in these canine cells and with testing whether the expression of telomerase genes is upregulated. Assume that you have access to both a small sample of healthy skin tissue from that founder dog and an unlimited supply of the immortal cells. Propose two complementary strategies (A, B) for estimating the number or length of telomeric repeats (using the methods covered in class or their extensions/variations) and two strategies for measuring telomerase expression levels (C, D).
(A) A hybridization-based quantitative DNA FISH approach (Q-FISH) – use a DNA or, ideally, a PNA probe (peptide nucleic acid, https://molecularcytogenetics.biomedcentral.com/articles/10.1186/1755-8166-6-42 ) complementary to the telomeric repeat and tagged with a fluorescent dye to detect the telomeres in metaphase spreads using fluorescence microscopy. The brightness of the fluorescent label hybridizing to the chromosomal ends will reflect the number of the repeats. You can compare the fluorescence intensity between the healthy sample and the immortal cell line sample using imaging software packages such as Image J.
(B) A qPCR-based approach – design primers for the telomeric repeats and for a single-copy gene in the genome as a normalization control. Telomeric primers have multiple binding sites (and hence will produce a smear of fragments of different sizes) but will show different levels of amplification (and hence of SYBR green or TaqMan fluorescence) from genomic DNA obtained from healthy tissue versus the cancer cell line. The telomeric repeat amplification data need to be normalized against qPCR data obtained for a control genomic locus (present in the genome only once) to control for different DNA inputs between samples. The design of PCR primers is critical, with intentional mismatches introduced into primers to prevent self-annealing of the forward and reverse primers (aka primer-dimer formation). One such clever primer design is presented here: https://www-ncbi-nlm-nih-gov.prox.lib.ncsu.edu/pmc/articles/PMC115301/ (see fig 1).
Other approaches, e.g. Telomere Restriction Fragment Analysis (a Southern blot with genomic DNA enzymatically digested by restriction enzymes, typically a mix of 4-cutters that do not recognize the telomeric repeats but degrade the rest of chromosomal DNA, and with the surviving telomeres detected using radioactively or fluorescently labelled telomeric probes) are acceptable for parts A and B.
(C) Telomerase expression can be analyzed at the RNA level for all three components (TERC, TERT and Dyskerin), e.g. reverse transcription of total RNA into cDNA using oligo-dT followed by qRT-PCR or ddPCR using gene-specific primer pairs and SYBR Green chemistry (or TaqMan probes). Again, the fluorescence/expression levels detected with gene-specific telomerase primers in the healthy tissue and the immortal cell line need to be compared and normalized against that of a control housekeeping gene like Actin.
(D) TERT and Dyskerin proteins in the healthy tissue versus the cell line can be analyzed by separating boiled tissue lysates by SDS-PAGE and detecting TERT and Dyskerin by Western blots using commercial primary and secondary antibodies. (TERC is not translated and is not relevant here). Some regions of these proteins are very conserved, so human and mouse antibodies should work well for dog samples (see, for example, https://www.labome.com/product/LifeSpan-Biosciences/LS-C110511.html) in combination with anti-human and anti-mouse secondary antibodies. To control for sample loading and allow for data normalization, an anti-ACTIN (or another housekeeping protein) primary antibody should be used in parallel. Secondary antibodies are conjugated to an enzyme or a fluorescent dye, enabling histochemical or fluorescent detection of TERT and Dyskerin.
Other methods, e.g., immunolocalization of TERT and Dyskerin or Northern blots of TERC, TERT and Dyskerin, are also acceptable gene expression evaluation techniques for parts C and D. Microarrays and RNA-seq are a poor choice for detecting the expression of just three genes. Various techniques based on TRAP (Telomerase Repeated Amplification Protocol) are also not a good choice for this question, as you were specifically asked to measure the expression rather than the activity of telomerase.
31
A form of naturally found dsDNA that is NOT right-handed
Z-DNA
32
A eukaryotic endonucleolytic enzyme that removes an RNA primer after nick-translation by the replicative DNA polymerase delta
FEN1 (+ DNA2)
33
A generic name of a molecular method to study protein-protein interactions in vivo by the reconstitution of fluorescent proteins separated into two halves; does NOT leverage yeast
BiFC
34
An essential protein complex in eukaryotic DNA replication that abbreviates the subunit names to their gene numbers in Japanese
GINS
35
A scientific discovery of certain nucleotide ratios in dsDNA that allowed Watson and Crick to infer specific nucleotide base-pairing
Chargaff’s rule
36
An enzymatic activity of DNA polymerases that increases the fidelity of DNA synthesis (be sure to indicate its directionality!)
3’-5’ exonuclease
37
A generic name of an enzyme that synthesizes short stretches of RNA in DNA replication
Primase
38
An alternative secondary structure in DNA that is the culprit of repeat expansions in several human neurodegenerative disorders and muscular myopathies
Slipped structure
39
A version of Cas9 that has one of its nuclease domains disabled via a mutation
Nickase (D10A)
40
An amplification-based method of quantifying gene expression that relies on a displacement and degradation of an internal labelled probe during DNA synthesis
TaqMan RT-qPCR
41
A DNA-binding bacterial protein that can block the progression of a replication fork if it is approached from its non-permissive side
Tus
42
A method of interrogating interactions between a protein of interest and a piece of labelled DNA on a polyacrylamide gel; does NOT require the addition of any enzymes
EMSA
43
A sequence property of all prokaryotic and eukaryotic origins of replication
AT-rich
44
A protein-denaturing detergent utilized in most plasmid DNA extraction protocols
SDS
45
A phenomenon of transmitting energy between two juxtaposed reporter proteins
FRET
46
A display of hundreds of tiny droplets of different recombinant proteins on a solid surface
Functional protein microarray
47
A plant sterol-inspired synthetic nucleotide label used in RNA in situ hybridization
Digoxygenin
48
A prevalent method of clonal amplification of DNA on primer-coated beads in several next-gen sequencing approaches
Emulsion PCR
49
Treated bacterial cells made capable of accepting foreign DNA upon heat shock
Chemically competent cells
50
A short nucleotide sequence that allows Cas9 to distinguish between its own CRISPR locus and invader’s DNA
PAM
51
A bacterial species used to first demonstrate the nature of an organic polymer that can convert a non-pathogenic strain to a pathogenic strain
Streptococcus pneumoniae
52
A long-range 3rd generation sequencing technology that can directly sequence RNA
Oxford nanopore
53
DNA synthesis biproducts detected by Ion Torrent semiconductor sequencing
H+ (protons)
54
A generic name of a bioluminescent reporter that works in the presence of a chemical substrate and ATP
luciferase
55
A genome-wide protein-DNA interaction method that can differentiate between protein binding to methylated and unmethylated DNA
DAP-seq
56
A method of circular DNA replication that does NOT produce a replication bubble
Rolling circle replication
57
What structural feature in the B-DNA double helix is determined by the relative angle of the glycosidic bonds (that connect deoxyribose with a base) in a base pair?
The uneven size of the major and minor groove of DNA
58
A next-gen DNA sequencing technology that relies on the ligation of sets of labelled interrogation probes to infer the DNA sequence
SOLiD
59
A hexameric donut-shaped protein complex that separates the strands of double-stranded DNA in eukaryotes enabling replication forks to progress
MCM2-7
60
An enzyme in E. coli replication that removes RNA primers and fills in the resulting gaps in DNA
DNA Pol I
61
A 3rd generation DNA sequencing method that interprets disruptions in electric current flowing through a protein channel when DNA molecule is being threaded through its opening
Oxford nanopore
62
A protein-protein interaction interrogation technique that relies on the use of a split transcription factor in yeast
Yeast 2 hybrid
63
Upon cyclin-mediated activation, it phosphorylates multiple eukaryotic replication initiation factors enabling S-phase of the interphase to begin
S-phase CDK/ DDK
64
An original name given by scientists to the organic material from dead virulent Pneumococcal cells that converted a non-virulent strain to a virulent strain
Transforming principle
65
A whole-genome microarray that contains oligos representing an entire genome of a species of interest
Tiling microarray
66
A hexameric protein complex in mammals that protects chromosomal ends, assists in T-loop formation
Shelterin
67
A reporter construct type that links a promoter of a gene of interest to a fluorescent, luminescent, or histochemical marker
Transcriptional reporter
68
A replication initiator protein in E. coli that binds to multiple sites in the oriC throughout the cell cycle
DnaA
69
A laboratory method that separates proteins on a gel based on their size
SDS-PAGE
70
A traditional nucleic acid staining dye used in DNA and RNA electrophoresis that is being replaced in some labs due to safety concerns
Ethidium Bromide
71
A restriction enzyme utilized in the construction of TALENs and Zinc Finger Nucleases
Fok1
72
A generic sequence name for a short DNA element found next to the gRNA target site in CRISPR/Cas technologies; required for DNA cleavage
PAM
73
A Cas protein that targets RNA
Cas13
74
Bacterial cells capable of accepting foreign DNA upon brief heat shock
Chemically competent cells
75
A state-of-the-art version of CRISPR/Cas9 genome editing that leverages reverse transcription of a modified gRNA to introduce DNA changes at the cut site
Prime editing
76
Molecular basis of quenching fluorescence of a dye in a TaqMan probe
FRET
77
The main replicative DNA polymerase in bacteria
DNA polymerase III
78
A protein-RNA interaction detection method that involves UV crosslinking and the use of antibodies
CLIP
79
A name of a duo of amino acids in a TALE repeat that dictate the nucleotide that the repeat will recognize
RVD
80
B-DNA handedness
right
81
The end in the DNA or RNA that carries the phosphate group
5'
82
A general name for DNA base pairs that form in H-DNA alongside with classical Watson-Crick base pairs to accommodate a 3rd strand of DNA in the major groove of the DNA double helix
Hoogsteen base pairs
83
A plant steroid leveraged in in situ hybridization probes that is covalently linked to uridines
Digoxygenin
84
A reverse transcriptase component of telomerase in eukaryotes
TERT
85
Directionality of nick translation
5’ to 3’
86
annotate a yeast 3 hybrid scheme (2023 question 3)
A – a DNA cis-element (e.g., a LexA operator sequence) in a promoter recognized in a sequence-specific manner by the DNA binding domain C
B – a reporter gene, such as an auxotrophic or colorimetric marker (e.g., His3 or LacZ), that enables selection of yeast colonies that successfully assembled the triple hybrid and reconstituted the split transcription factor
C – a DNA binding domain (e.g., that of LexA) that is recruited to the DNA cis-element in A
D – a well-characterized RNA-binding domain, such as that of an MS2 coat protein, that binds to RNA E
E – an RNA aptamer (e.g., MS2) recognized by the RNA-binding domain D
F – an RNA molecule (bait/prey) that is being tested for the interaction with a putative RNA-binding protein G (prey/bait)
G – an RNA binding protein (prey/bait) that is being tested for the interaction with RNA F (bait/prey)
H – a transcription activation domain (e.g., that of GAL4) that helps recruit RNA Pol II and initiate transcription
87
List any four of the six mechanisms discussed in class that prevent DNA over-replication in E. coli
*Poor binding of the replication initiator protein DnaA to the hemimethylated OriC (whereas DnaA binding to high-, medium- and low-affinity DnaA boxes in the DNA is a prerequisite of replication initiation)
*Strong binding of the replication repressor protein SeqA to the hemimethylated OriC
*Transcriptional repression of the DnaA gene due to the binding of SeqA to its promoter
*Transcriptional repression of the DnaA gene due to the binding of DnaA itself to its own promoter
*Predominance of the inactive DnaA-ADP protein (as opposed to active DnaA-ADP) in the cell
*Doubling in the number of high-affinity DnaA boxes in the duplicated genome that sequester DnaA
*Ter sites bound by TUS proteins ensure unidirectional progression of replication forks, ensuring that replication cannot continue past the Ter region
88
What types of secondary structures may form in the DNA if the primary DNA sequence contains direct tandem repeats?
Slipped structures
89
slipped structures may form in the DNA if the primary DNA sequence contains direct tandem repeats,
what can happen to those repeats over time?
They expand (or shrink) in copy number
(alternative answer: forward and backward slippage)
90
Slipped structures may form in the DNA if the primary DNA sequence contains direct tandem repeats,
over time these repeats expand or shrink in copy number. What two molecular processes makes these changes possible?
DNA replication and DNA repair
91
List an example of a human disorder that is thought to be a result of direct tandem repeats/slipped structures
Huntington’s disease
92
What is the optimal application to: Test for binding between a purified recombinant mouse protein and an end-labeled promoter fragment in vitro and pinpoint the DNA element(s) bound
DNase I footprinting
93
What is the optimal application to: Determine tissue-specific expression patterns of four genes of interest in fixed human liver slices
In-situ hybridization
translational reporter (only if reporter is compatible with tissue fixation)
94
What is the optimal application to: Investigate the possibility of a Drosophila transcription factor interacting with a promoter element of interest in a heterologous system in vivo
Yeast one hybrid
95
What is the optimal application to: Establish the genome-wide pattern of DNA binding for a maize transcription factor of interest
ChIP-seq
96
What is the optimal application to: Test for the interaction between two C. elegans proteins of interest in a native system in vivo using two-color fluorescence microscopy
FRET
97
What is the optimal application to: Convert tiger mRNA into cDNA
reverse transcription
98
What is the optimal application to: Compare transcript abundance of 100 selected genes across 10 tumor samples
nanostrings
99
What is the optimal application to: Visualize expression of a tagged protein of interest in the development of a transgenic chicken
translational reporter
westernblot
100
What is the optimal application to: Confirm lack of point mutations in a construct of interest
sanger sequencing
101
What is the optimal application to: Evaluate the effect of salt stress on the expression of two Arabidopsis proteins of interest over time using specific antibodies
western blot
102
What is the optimal application to: Generate a knockout allele of a gene of interest in a human cell line
zinc finger nuclease
(ZFN was answer in from pre-determined list but crispr would work too)
103
What is the optimal application to: Identify polymorphic sites in the genomes of 10 wild-caught snakes
Illumina sequencing
104
State the names of the techniques that would enable you to: Amplify a gene of interest from chicken genomic DNA and incorporate restriction sites of interest at its flanks to prepare the DNA fragment for subcloning into a plasmid
Polymerase chain reaction (PCR) with primers containing restriction enzyme sites
105
State the names of the techniques that would enable you to: Introduce a DNA plasmid into electro-competent E. coli cells using a pulse of electricity
Electroporation
106
State the names of the techniques that would enable you to: Confirm identity of the construct your colleague shared with you if you do NOT have primers specific for that plasmid but know its sequence
Restriction digest and agarose gel electrophoresis
107
State the names of the techniques that would enable you to: Determine [with the help of commercial antibodies] if a yeast strain you transformed with the plasmid of interest expresses the tagged recombinant protein
SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis) and Western blotting, or immunolocalization/immunocytochemistry
108
State the names of the techniques that would enable you to: Purify the recombinant protein of interest from a cellular lysate taking advantage of the tag the protein harbors
Affinity purification/immuno-precipitation
109
State the names of the techniques that would enable you to: Establish the identity of the proteins that co-purify with the recombinant protein of interest (assume no prior knowledge of the interactors)
Co-immunoprecipitation or pull-down followed by Mass Spectrometry
110
State the names of the techniques that would enable you to: Confirm the interaction between your protein of interest and its putative partner protein in vivo using one-color fluorescence microscopy
Bimolecular fluorescence complementation
111
State the names of the techniques that would enable you to: Generate a targeted knockout of the gene of interest in vivo
CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeat/ CRISPR-associated9)
112
State the names of the techniques that would enable you to: Confirm that the gene of interest was successfully disrupted and identify the specific molecular lesion within the gene (note that multiple steps may be required)
Genomic DNA extraction, PCR amplification with primers flanking the edit site, and Sanger sequencing of the PCR fragment
113
State the names of the techniques that would enable you to: Characterize the effect of the newly made loss-of-function mutant in terms of global gene expression misregulation that the mutation has caused
Ribonucleic acid (RNA)-seq (e.g., via Illumina sequencing)
114
State the names of the techniques that would enable you to: Confirm expression changes of a handful of genes (identified as misregulated in the mutant in part J) by another approach
Reverse transcription followed by quantitative real-time PCR (qRT-PCR) or digital droplet PCR (ddPCR); in situ hybridization, transcriptional reporters
115
