End-of-chapter questions below · Part 1 of 2 · 10 questions per part
Part 1 of 2
Cell Fractionation, Nucleic Acid Analysis, and DNA Technology
Modern cell biology is inseparable from the techniques used to study it — every major discovery, from the structure of the ribosome to the function of oncogenes, depended on developing the right tool to ask the right question.
Cell biologists use an enormous toolkit to dissect the molecular events inside living cells. The first challenge is often separating the cellular components of interest from everything else. Cell fractionation accomplishes this by breaking cells open (homogenization) and then using differential or density-gradient centrifugation to pellet organelles by size and density.
Nuclei pellet at low centrifugal forces (~600 × g), mitochondria and lysosomes at moderate forces (~10,000 × g), and ribosomes/microsomes at high forces (~100,000 × g). Density-gradient centrifugation (e.g., sucrose or cesium chloride gradients) achieves finer separations based on buoyant density.
Key term
Differential centrifugation
Sequential centrifugation at increasing speeds to separate subcellular fractions by size and density; each step pellets progressively smaller organelles.
Gel Electrophoresis, Western Blotting, and Microscopy
Gel electrophoresis separates molecules by size through a porous gel matrix under an electric field. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) denatures proteins and coats them with negative charge, so they migrate by size alone. Nucleic acids are run on agarose gels. After separation, specific proteins can be detected by Western blotting: transferring gel-separated proteins onto a membrane, then probing with a primary antibody against the protein of interest followed by an enzyme-linked secondary antibody for detection.
Light microscopy allows visualization of cells and their organelles down to the diffraction limit (~200 nm). Phase contrast and differential interference contrast (DIC) microscopy generate contrast without staining. Fluorescence microscopy uses fluorescent dyes or GFP-tagged proteins to image specific molecules in live cells.
PCR, DNA Cloning, Restriction Enzymes, and Sequencing
The polymerase chain reaction (PCR) amplifies specific DNA sequences exponentially using a thermostable DNA polymerase (Taq), two flanking primers, and cycles of denaturation, annealing, and extension. Starting from a single DNA molecule, PCR can generate billions of copies in hours.
Restriction enzymes (endonucleases) cut double-stranded DNA at specific sequences called restriction sites, producing defined fragments. When cut fragments are joined to a vector (plasmid, phage, or artificial chromosome) by DNA ligase, the result is recombinant DNA that can be introduced into bacterial host cells for amplification. DNA sequencing — originally by Sanger dideoxy chain-termination, now by massively parallel next-generation sequencing — determines the nucleotide order of any DNA fragment.
Key term
PCR (Polymerase Chain Reaction)
An in vitro technique that exponentially amplifies a specific DNA sequence using repeated cycles of heat denaturation, primer annealing, and DNA synthesis by thermostable polymerase.
Practice questions — Part 1Score: 0 / 10
1. In differential centrifugation, which organelle fraction is typically collected in the FIRST low-speed pellet (~600 × g)?
Nuclei are the largest and densest organelles and are pelleted at the first, lowest centrifugal force step (~600 × g). Subsequent steps at ~10,000 × g pellet mitochondria; ~100,000 × g pellets ribosomes and microsomes.
2. In SDS-PAGE, what does SDS (sodium dodecyl sulfate) do to proteins?
SDS is an anionic detergent that denatures proteins and binds proportionally to mass, giving all proteins a uniform negative charge density. This masks inherent charge differences so that migration through the gel matrix depends only on molecular size.
3. In PCR, what is the role of the annealing step?
The annealing step (~50–65°C, depending on primer Tm) allows the two single-stranded primers to base-pair with complementary sequences flanking the target region on the template strands.
4. Western blotting is used to detect:
Western blotting (immunoblotting) involves SDS-PAGE, transfer of proteins to a nitrocellulose or PVDF membrane, blocking, incubation with a protein-specific primary antibody, then an enzyme-conjugated secondary antibody for chemiluminescent or colorimetric detection.
5. Restriction enzymes recognize specific DNA sequences called restriction sites. What type of cuts do most restriction enzymes make?
Type II restriction endonucleases make double-stranded cuts within or near palindromic recognition sequences. Staggered cuts produce 5' or 3' single-stranded overhangs ("sticky ends"); blunt-end cutters produce flush ends, both useful for cloning.
6. Why is Taq polymerase used in PCR rather than E. coli DNA polymerase I?
Taq polymerase, isolated from the thermophilic bacterium Thermus aquaticus, withstands the ~95°C denaturation step without being inactivated. Mesophilic polymerases like E. coli Pol I would denature irreversibly in each cycle.
7. What technique uses labeled probes to detect specific RNA transcripts on a membrane after gel electrophoresis?
Northern blotting detects RNA: RNA is separated on a denaturing agarose gel, transferred to a membrane, and hybridized with a labeled DNA or RNA probe complementary to the target transcript. Southern blotting detects DNA; Western detects protein.
8. When a foreign DNA fragment is inserted into a plasmid vector and the recombinant plasmid is introduced into bacteria, what process allows the insert to be amplified?
Plasmid vectors contain an origin of replication recognized by bacterial replication machinery. Each time a bacterium divides, the plasmid — along with its cloned insert — is replicated and distributed to daughter cells, amplifying the insert.
9. The Sanger (dideoxy chain-termination) sequencing method relies on:
Dideoxynucleotides (ddNTPs) lack a 3'-OH, so their incorporation terminates chain elongation. Using four ddNTPs (each labeled with a different fluorescent dye) generates a nested set of fragments that, when separated by capillary electrophoresis, reveals the sequence.
10. Which technique is used to separate cell organelles based on their buoyant density in a continuous gradient?
In equilibrium (isopycnic) density-gradient centrifugation (e.g., CsCl or sucrose gradient), particles migrate until they reach the position in the gradient matching their own buoyant density — ideal for separating organelles with similar sizes but different densities.
0/10
Part 1 complete
End-of-Part 1 Questions
Type your answer, then click Check answer for feedback and a sample answer.
Section B — Recall Questions
B1
Describe the principle behind differential centrifugation and how it separates cellular components.
Sample answer: Cells are homogenized and the lysate is centrifuged at progressively increasing speeds. At each speed, a pellet is collected containing organelles too dense or too large to remain suspended. Nuclei pellet at low speed (~600 × g), mitochondria at moderate speed (~10,000 × g), and ribosomes/microsomes at high speed (~100,000 × g), allowing sequential isolation of each fraction.
B2
Why does SDS-PAGE separate proteins by molecular weight rather than by charge or shape?
Sample answer: SDS denatures proteins and binds uniformly to hydrophobic regions at approximately 1.4 g SDS per g protein, giving all proteins the same negative charge-to-mass ratio. With inherent charge differences masked, migration through the polyacrylamide gel depends only on molecular weight — smaller proteins migrate faster.
B3
What are the three steps of one PCR cycle, and what happens at each temperature?
Sample answer: (1) Denaturation (~95°C): heat separates the double-stranded DNA into single strands. (2) Annealing (~50–65°C): primers hybridize to complementary sequences flanking the target. (3) Extension (~72°C): Taq polymerase synthesizes new DNA strands from each primer, doubling the number of target molecules.
B4
Explain how restriction enzymes and DNA ligase are used together to create recombinant DNA.
Sample answer: The same restriction enzyme cuts both the vector and the insert DNA, generating complementary sticky (or blunt) ends. When mixed, complementary sticky ends can base-pair. DNA ligase then covalently seals the phosphodiester backbone, creating recombinant DNA in which the insert is stably incorporated into the vector.
B5
Outline the steps of a Western blot experiment.
Sample answer: (1) Lyse cells and separate proteins by SDS-PAGE. (2) Transfer proteins from gel to nitrocellulose or PVDF membrane (electroblotting). (3) Block non-specific binding with BSA or milk. (4) Incubate with primary antibody specific to target protein. (5) Wash, then incubate with enzyme-conjugated secondary antibody. (6) Detect with chemiluminescent substrate; bands appear where target protein is present.
B6
How is GFP used to study protein localization in living cells by fluorescence microscopy?
Sample answer: The gene encoding GFP is fused in-frame to the gene of interest, creating a GFP-tagged fusion protein expressed in cells. When illuminated at the excitation wavelength (~488 nm for EGFP), GFP emits green fluorescence, revealing where the tagged protein is located in real time without killing the cell.
B7
Why do dideoxynucleotides (ddNTPs) terminate DNA chain elongation in Sanger sequencing?
Sample answer: ddNTPs lack a 3'-hydroxyl group. When incorporated into a growing DNA chain, there is no 3'-OH for the next nucleotide to be added, so elongation stops. Each of the four ddNTPs (ddATP, ddCTP, ddGTP, ddTTP) terminates synthesis at a different base position.
B8
How do researchers confirm that bacteria have taken up a recombinant plasmid rather than a re-circularized empty vector?
Sample answer: Classic approach: blue-white selection. The insert is cloned into a multiple cloning site within the lacZ gene on the vector. Bacteria with an empty vector produce functional β-galactosidase (blue colonies on X-gal plates); bacteria with an insert have disrupted lacZ and form white colonies. Positive clones are confirmed by colony PCR or restriction digest analysis.
B9
Why is phase contrast microscopy useful for viewing living, unstained cells?
Sample answer: Phase contrast optics convert small differences in refractive index (caused by differences in density of cellular structures) into differences in light intensity. This generates contrast in transparent, unstained living cells without requiring toxic dyes or fixation, allowing observation of cell morphology and organelle dynamics in real time.
B10
What determines the migration rate of a DNA fragment in agarose gel electrophoresis?
Sample answer: DNA is uniformly negatively charged (one phosphate per base), so in an electric field all DNA migrates toward the positive electrode. Migration rate is inversely proportional to fragment size (in base pairs) because smaller fragments navigate the gel matrix pores more easily. Fragment sizes are estimated by comparison to a DNA ladder of known sizes.
Section C — Critical Thinking Questions
C1
PCR is extraordinarily sensitive, capable of amplifying a single molecule of DNA. How might this sensitivity be a disadvantage in diagnostic or forensic applications?
Sample answer: Extreme sensitivity makes PCR highly susceptible to contamination. A single stray DNA molecule — from a previous reaction, a researcher's skin cells, or environmental sources — can be amplified and generate a false positive result. Strict laboratory protocols (separate pre- and post-PCR areas, negative controls, UV decontamination) are essential to prevent this.
C2
Next-generation sequencing (NGS) has essentially replaced Sanger sequencing for whole-genome projects. What key advantage does NGS have over Sanger sequencing?
Sample answer: NGS performs massively parallel sequencing — millions or billions of short reads are generated simultaneously from a single library preparation. This makes it orders of magnitude faster and cheaper per base than Sanger sequencing (which sequences one fragment at a time). A human genome can be sequenced in days for ~$1,000 by NGS versus years and billions of dollars by Sanger.
C3
Human insulin for diabetic patients was historically extracted from pig or cow pancreases. How did recombinant DNA technology improve insulin production?
Sample answer: The human insulin gene was cloned into expression vectors and introduced into E. coli or yeast cells, which produce large quantities of human insulin identical to the endogenous protein. This eliminated supply limitations and animal-derived contaminants, reduced the risk of immune reactions to non-human insulin sequences, and dramatically lowered production costs.
C4
When using differential centrifugation to purify mitochondria, why might the mitochondrial fraction be contaminated with lysosomes, and how would you address this?
Sample answer: Mitochondria and lysosomes have overlapping sedimentation rates at ~10,000 × g. To separate them, the crude pellet is resuspended and run on a sucrose density gradient (e.g., equilibrium isopycnic gradient). Mitochondria (~1.19 g/mL) and lysosomes (~1.21 g/mL) have slightly different buoyant densities and will band at different positions in the gradient, allowing their separation.
C5
A researcher tags a protein with GFP and observes it localizing to the nucleus. However, a colleague argues that this localization might be artifactual. What potential artifacts could arise from GFP tagging, and how would you validate the result?
Sample answer: Artifacts include: (1) GFP bulk may misfold or mask targeting signals, altering localization; (2) overexpression from a strong promoter may saturate normal targeting machinery; (3) GFP dimerizes weakly at high concentrations, possibly clustering the fusion protein. Validation: confirm with antibody staining of endogenous protein; use a smaller tag (e.g., HA, FLAG); express at physiological levels using the endogenous promoter (knock-in); use CRISPR to tag the endogenous locus.
Section D — Interactive Questions
D1
What thermostable enzyme is used to synthesize new DNA strands in PCR?
D2
What blotting technique uses antibodies to detect a specific protein after SDS-PAGE?
D3
What enzyme joins a DNA insert to a vector backbone during cloning?
D4
What fluorescent protein is most commonly used to tag proteins and study their localization in living cells? (abbreviation)
D5
What type of centrifugation separates organelles based on buoyant density in a sucrose or CsCl gradient?
Part 2 →
Having covered nucleic acid-level tools, we now turn to protein analysis, genome editing, and systems biology — technologies including mass spectrometry, CRISPR-Cas9, RNA-seq, and computational approaches that allow us to study the cell as an integrated system.
Part 2 of 2
Protein Analysis, Genome Editing, and Systems Biology
Once proteins are purified and identified, their functions and interactions can be probed with a growing toolkit. Protein purification exploits properties such as size (gel filtration chromatography), charge (ion-exchange chromatography), hydrophobicity (hydrophobic interaction chromatography), and specific binding affinity (affinity chromatography). His-tagged proteins, for example, can be purified in a single step using nickel-chelate affinity resin.
Mass Spectrometry and Protein Interactions
Mass spectrometry (MS) identifies proteins by measuring the mass-to-charge ratio (m/z) of peptides generated by proteolytic digestion. In a typical "shotgun proteomics" workflow, a complex protein mixture is digested with trypsin, peptides are separated by liquid chromatography, and fragmentation spectra are searched against protein databases to identify each protein. This approach can identify thousands of proteins in a single experiment.
Protein-protein interactions can be detected by co-immunoprecipitation (co-IP), yeast two-hybrid assays, or proximity ligation assays (PLA). Immunoprecipitation (IP) uses a specific antibody to pull down the target protein, and its interaction partners are then identified by MS or Western blot.
Key term
CRISPR-Cas9
A bacterial adaptive immune system repurposed as a programmable genome editing tool. A guide RNA directs the Cas9 nuclease to cut double-stranded DNA at a specific genomic sequence, enabling gene knockout, correction, or insertion.
CRISPR-Cas9, RNA-seq, and Systems Biology
CRISPR-Cas9 has revolutionized genome editing. A ~20 nt single guide RNA (sgRNA) directs Cas9 to cut the genome adjacent to a protospacer adjacent motif (PAM). The resulting double-strand break is repaired either by error-prone NHEJ (causing insertions/deletions that knockout the gene) or by homology-directed repair using a provided template (enabling precise corrections or insertions).
RNA-seq (RNA sequencing) provides a transcriptome-wide snapshot of gene expression by converting all mRNA in a sample to cDNA and sequencing millions of fragments. Reads are mapped to a reference genome; read counts per gene reflect expression levels, enabling comparison between conditions or cell types with unprecedented sensitivity.
Proteomics and bioinformatics together underpin systems biology — the integrative analysis of all molecular components and their interactions in a cell. Network models of protein-protein interactions, metabolic pathways, and gene regulatory circuits allow predictions about how perturbations (mutations, drugs) will affect cellular behavior.
Practice questions — Part 2Score: 0 / 10
1. In CRISPR-Cas9 genome editing, what component determines which genomic sequence is cut?
The ~20 nt spacer sequence within the sgRNA base-pairs with the complementary genomic DNA strand, directing Cas9 to that specific location. Cas9 itself provides the nuclease activity but has no intrinsic DNA sequence preference.
2. After Cas9 creates a double-strand break, what repair pathway most commonly creates gene knockouts?
NHEJ is the predominant DSB repair pathway in most eukaryotic cells. It rejoins broken ends without a template, frequently introducing small insertions or deletions (indels) at the cut site. If these indels fall in a coding exon, they often cause frameshifts and loss of protein function.
3. In a typical RNA-seq experiment, what is the first step performed on the isolated mRNA?
Standard RNA-seq converts mRNA to double-stranded cDNA using reverse transcriptase and then DNA polymerase. The cDNA library is then fragmented, adapter-ligated, and sequenced by next-generation sequencing. (Nanopore direct RNA sequencing is an emerging alternative.)
4. Affinity chromatography purifies proteins by:
Affinity chromatography exploits a specific binding interaction (e.g., His-tag/nickel, antibody/antigen, enzyme/substrate analog). The target protein binds the ligand on the column while other proteins flow through; the target is then eluted by adding a competing ligand or changing conditions.
5. Mass spectrometry in proteomics identifies proteins by measuring:
In MS-based proteomics, proteins are digested into peptides, ionized, and separated in a mass analyzer by m/z ratio. Tandem MS (MS/MS) fragments peptide ions; the resulting spectra are searched against databases (e.g., UniProt) to identify protein sequences.
6. Which of the following best describes a transgenic organism?
Transgenic organisms carry a foreign gene (transgene) stably integrated into their genome. Classic examples include transgenic mice expressing human disease genes for disease modeling, and transgenic crops expressing insecticidal Bt protein.
7. Co-immunoprecipitation (co-IP) is used to study:
In co-IP, a cell lysate is incubated with an antibody against protein A; the immune complex is precipitated. If protein B is a binding partner of protein A, it will co-precipitate and can be detected by Western blot or MS.
8. Systems biology differs from traditional reductionist biology in that it:
Systems biology integrates high-throughput datasets (transcriptomics, proteomics, metabolomics) with mathematical models to understand emergent properties of biological networks that cannot be predicted by studying individual components in isolation.
9. In the yeast two-hybrid assay, an interaction between two proteins is detected by:
In yeast two-hybrid, protein X is fused to the DNA-binding domain of a transcription factor, protein Y to the activation domain. Interaction between X and Y brings the two domains together, reconstituting the transcription factor and activating a reporter gene (e.g., HIS3 or lacZ).
10. What is a key advantage of CRISPR-Cas9 over earlier genome editing tools such as zinc finger nucleases (ZFNs) and TALENs?
ZFNs and TALENs require engineering new proteins for each target sequence, which is time-consuming and expensive. CRISPR-Cas9 uses a short, easily synthesized sgRNA to program specificity; changing the target requires only changing the ~20 nt spacer sequence, making it dramatically faster and cheaper to design.
0/10
Part 2 complete
End-of-Part 2 Questions
Type your answer, then click Check answer for feedback and a sample answer.
Section B — Recall Questions
B1
Describe how CRISPR-Cas9 creates a targeted double-strand break in genomic DNA.
Sample answer: A synthetic sgRNA containing a ~20 nt spacer complementary to the target sequence base-pairs with the genomic DNA adjacent to a PAM sequence (NGG for SpCas9). The Cas9 protein binds the sgRNA and uses two nuclease domains (RuvC and HNH) to cut each strand of the DNA duplex, creating a blunt double-strand break 3 bp upstream of the PAM.
B2
Outline the main steps of an RNA-seq experiment from cell lysis to data output.
Sample answer: (1) Lyse cells and isolate total RNA. (2) Enrich mRNA (poly-A selection or rRNA depletion). (3) Fragment RNA and reverse-transcribe to cDNA. (4) Ligate sequencing adapters to create a library. (5) Sequence millions of cDNA fragments by NGS. (6) Align reads to a reference genome or transcriptome; count reads per gene to quantify expression levels. Differential expression analysis compares counts between conditions.
B3
How does shotgun mass spectrometry proteomics identify proteins in a complex mixture?
Sample answer: The protein mixture is digested with trypsin into peptides, separated by liquid chromatography, then introduced into the mass spectrometer. Each peptide is fragmented (MS/MS) and the mass spectrum is searched against a protein sequence database to identify the peptide sequence and thus the parent protein.
B4
Explain how a His-tagged recombinant protein is purified by affinity chromatography.
Sample answer: A 6×His tag is genetically fused to the protein of interest. The cell lysate is applied to a nickel-nitrilotriacetic acid (Ni-NTA) resin. The His tag chelates the nickel ions, binding the target protein; other proteins flow through. The His-tagged protein is then eluted by adding imidazole (which competes for nickel binding) or lowering the pH.
B5
Describe the co-immunoprecipitation (co-IP) technique and what it is used to determine.
Sample answer: Cells are lysed under mild conditions to preserve protein complexes. The lysate is incubated with an antibody specific to protein A, then the antibody-antigen complex is precipitated (with protein A/G beads). The precipitate is washed and analyzed by Western blot or MS. If protein B co-precipitates, it physically interacts with protein A in the cell.
B6
What is a knockout mouse, and how has this model been used to study gene function?
Sample answer: A knockout mouse has one or both copies of a gene inactivated (often by homologous recombination or CRISPR-Cas9 in embryonic stem cells). By comparing knockout mice to wild-type littermates, researchers can determine the physiological function of the gene — what processes are disrupted, which tissues are affected, and whether the phenotype resembles a human disease.
B7
Why is bioinformatics essential for modern genomics and proteomics experiments?
Sample answer: Modern experiments generate terabytes of sequence data that cannot be analyzed manually. Bioinformatics tools align millions of sequencing reads to reference genomes (e.g., STAR, BWA), quantify gene expression, identify variants, annotate protein functions, and build interaction networks. Without computational pipelines, the data generated by NGS, MS-proteomics, and CRISPR screens would be uninterpretable.
B8
How can CRISPR-Cas9 be used to precisely correct a disease-causing point mutation (not just knockout the gene)?
Sample answer: In addition to sgRNA and Cas9, a donor DNA template carrying the corrected sequence is provided. After Cas9 cuts at the mutation site, homology-directed repair (HDR) uses the donor template to copy the correct sequence into the genome. This repairs the mutation precisely, rather than just disrupting the gene by NHEJ.
B9
What is two-dimensional (2D) gel electrophoresis and what advantage does it have over standard SDS-PAGE for proteomics?
Sample answer: 2D gel electrophoresis separates proteins first by isoelectric point (pI) in the first dimension (isoelectric focusing) and then by molecular weight in the second dimension (SDS-PAGE). This two-dimensional resolution separates thousands of proteins as individual spots that can be excised, digested, and identified by MS — far more proteins than a 1D SDS-PAGE gel can resolve.
B10
What is an "emergent property" in systems biology, and why can it not be predicted from studying components individually?
Sample answer: An emergent property arises from interactions among components of a system and cannot be predicted from the properties of any individual component alone. Example: oscillatory behavior in the cell cycle arises from feedback loops among cyclins, CDKs, and their inhibitors; no single protein oscillates independently. Network-level analysis and mathematical modeling are needed to understand and predict such behaviors.
Section C — Critical Thinking Questions
C1
CRISPR-Cas9 can cause "off-target" cuts at genomic sites similar (but not identical) to the intended target. Why is this a serious concern for therapeutic applications, and how are researchers addressing it?
Sample answer: Off-target cuts could disrupt tumor suppressor genes or essential genes, potentially causing cancer or cell death in a patient — unacceptable for clinical use. Approaches to minimize off-targets include: high-fidelity Cas9 variants (eSpCas9, HiFi Cas9) with reduced tolerance for mismatches; using paired nickases (Cas9-D10A) that create single-strand nicks (requiring two nearby guides for a DSB); and delivering Cas9 as a ribonucleoprotein (RNP) rather than a plasmid to reduce its active time in the cell.
C2
A researcher uses RNA-seq to compare gene expression in cancer cells versus normal cells and finds 500 differentially expressed genes. What statistical and biological considerations are important before concluding that any of these genes drives the cancer phenotype?
Sample answer: Statistical considerations: multiple testing correction (FDR/Benjamini-Hochberg) is essential — with 20,000 genes tested, many will appear significant by chance. Biological considerations: differential expression may be a consequence rather than a cause of cancer (passenger vs. driver). Validation by orthogonal methods (RT-qPCR, Western blot) is required. Functional experiments (knockdown, overexpression, CRISPR knockout) are needed to determine if a gene causally drives the cancer phenotype.
C3
Studies have shown that mRNA levels (measured by RNA-seq) often correlate poorly with protein levels (measured by proteomics) for the same gene. What biological mechanisms could explain this discordance?
Sample answer: Post-transcriptional regulation: miRNAs and RNA-binding proteins can repress translation without changing mRNA levels. Translational efficiency varies by mRNA (codon usage, 5' UTR secondary structure, ribosome availability). Post-translational modifications can alter protein stability or activity without affecting mRNA. Protein half-lives differ dramatically (minutes to days) while mRNA abundance changes faster. These layers of regulation mean mRNA is an imperfect proxy for protein abundance.
C4
In 2018, He Jiankui announced the birth of genome-edited human babies with CRISPR-Cas9 modifications to the CCR5 gene to confer HIV resistance. Why did the scientific community respond with widespread condemnation?
Sample answer: Key concerns: (1) Germline editing is heritable — any off-target effects would be passed to all descendants, with consequences unknown for generations. (2) The medical need was unproven — HIV transmission could have been prevented by other means. (3) Off-target cuts were not fully characterized; safety was not established. (4) The editing was done without proper regulatory approval, oversight, or genuine informed consent. (5) It set a precedent for "designer babies," raising profound ethical concerns about equity and enhancement.
C5
In a protein-protein interaction (PPI) network, "hub" proteins with many interaction partners are often essential for cell survival. How might this knowledge guide drug discovery efforts?
Sample answer: Hub proteins are vulnerable nodes: disrupting them affects many downstream processes. If a hub is specifically overactive in cancer cells (e.g., Myc, p53), it becomes an attractive drug target — inhibiting it could collapse multiple oncogenic pathways simultaneously. However, hubs in normal cells are also often essential, so drugs targeting them may cause toxicity. This drives interest in targeting hub-specific protein-protein interactions with small molecules rather than blocking the hub's enzymatic activity broadly.
Section D — Interactive Questions
D1
What programmable genome editing system uses a guide RNA and a nuclease to cut DNA at a specific sequence? (abbreviation)
D2
What sequencing approach converts mRNA to cDNA and quantifies transcriptome-wide expression? (two words, hyphenated)
D3
What instrument identifies proteins by measuring the mass-to-charge ratio of peptide fragments? (two words)
D4
What type of chromatography purifies a His-tagged protein using nickel resin?
D5
What error-prone DNA repair pathway introduces indels at a CRISPR-Cas9 cut site to create gene knockouts? (abbreviation)