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Questions and Answers
What determines antigen binding specificity in monoclonal antibodies?
What is the primary advantage of monoclonal antibodies over polyclonal antibodies?
During the hybridoma creation process, what is used to select for desired antibody-producing cells?
What is the main purpose of sequence similarity searching?
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What defines orthologs in the context of genetic sequences?
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What does 'identity' refer to when comparing sequences?
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Which method provides a global alignment of sequences?
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What major achievement did Milstein and Köhler contribute to in the 1970s?
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What is the main purpose of the Cas9 enzyme in the CRISPR-Cas9 system?
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Which method of gene editing is more efficient for specific modifications but has limitations?
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What is a potential drawback of using primary cells for gene editing?
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What challenge in gene manipulation is highlighted by the possibility of varying phenotypes?
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Which of the following best describes the CRISPRa technique?
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What factors influence the choice of gene editing technique?
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What is a significant concern related to CRISPR technology in gene editing?
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Which characteristic is NOT typically associated with immortalized cells?
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What is the main advantage of using electron microscopy over light microscopy?
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What is the maximum resolution achievable with a Transmission Electron Microscope (TEM)?
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Which type of electron microscope is primarily used to examine surface details?
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What technique is commonly used for relative quantification in proteomics?
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In a TEM, what happens to the sample that might affect imaging?
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What is a key element of the Cryo-EM workflow?
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Which of the following is NOT a common post-translational modification (PTM)?
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In clinical diagnostics, proteomics is primarily used for what purpose?
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Which method of gene manipulation results in permanent changes to gene expression?
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What is a significant challenge in proteomics related to data analysis?
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What is one major concern when using RNA interference (RNAi) to knock down gene expression?
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What role do post-translational modifications (PTMs) serve in proteins?
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When developing disease models, why is gene manipulation important?
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What is a benefit of identifying mutations in diseases?
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Which type of genetic variation commonly leads to conditions like Down syndrome?
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What is the primary focus of the Candidate Gene Approach in identifying mutations?
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Which of the following best describes complex disorders?
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What is the primary purpose of Genome-Wide Association Studies (GWAS)?
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Which of the following would NOT be considered a structural variation?
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In which inheritance pattern are traits carried on non-sex chromosomes?
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Which method is considered the most comprehensive but also the most expensive for detecting genetic variants?
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Study Notes
Monoclonal Antibodies
- Monoclonal antibodies (mAbs) are produced by a single B cell clone and bind to one specific epitope.
- Hybridoma technology involves fusing B cells with myeloma cells to create hybridomas that produce antibodies indefinitely.
- Advantages of mAbs over polyclonal antibodies include high epitope specificity, genetic manipulability (humanized antibodies), and almost infinite production.
Production Process
- Clonal selection involves selecting B cells specific to an antigen and immortalizing them by fusion with myeloma cells.
- HAT selection is used to kill unwanted cells and select for hybridomas.
- Supernatants from cultured cells are screened for antibody production, and stable clones are expanded and preserved.
Applications
- mAbs are used for affinity purification of specific antigens, immunohistochemistry to locate specific cells in tissues, enzyme-linked immunosorbent assay (ELISA) to quantify specific proteins, and Western blotting to detect specific proteins in samples.
Historical Context
- The development of monoclonal antibodies based on immune specificity and clonal selection earned Milstein and Köhler the Nobel Prize in 1984.
- They pioneered hybridoma technology in the 1970s.
Introduction to Sequence Similarity Searching
- This method compares a query sequence (DNA, RNA, or protein) to a database of known sequences to find regions of similarity.
- It is used to identify homologous sequences, infer functional relationships, detect evolutionary connections, and annotate genes or proteins.
Key Concepts
- Homology refers to similarity due to shared ancestry.
- Orthologs are genes in different species from a common ancestor that typically perform the same function.
- Paralogs are genes within the same species that arose by duplication and may have new functions.
- Similarity considers conservative substitutions between sequences, while identity requires exact matching of residues.
Types of Searches
- Global alignment compares sequences across their entire length using algorithms like Needleman-Wunsch.
Quantification in Proteomics
- Relative quantification compares protein abundance between samples using techniques like 2D gel electrophoresis or chemical labeling (iTRAQ, TMT).
- Absolute quantification determines exact protein quantities using internal standards or label-free approaches.
Post-Translational Modifications (PTMs)
- Common PTMs include phosphorylation, glycosylation, ubiquitination, acetylation, and methylation.
- PTMs regulate protein function, interactions, and localization.
Applications of Proteomics
- It has applications in clinical diagnostics (identifying disease biomarkers), drug development (discovering protein targets), and molecular network discovery (understanding protein-protein interactions and signaling pathways).
Challenges and Considerations
- Proteomic data is complex and requires advanced analysis tools.
- False discovery rate (FDR) is used to assess the accuracy of protein identifications from MS data.
Why Manipulate Gene Expression?
- Investigating gene function involves reducing or increasing gene expression to understand its role in biological processes.
- Loss of function (LOF) techniques like RNA interference reduce gene expression.
- Gain of function (GOF) involves overexpressing genes to observe phenotypic changes.
- Disease models are created to study disease mechanisms.
- Modifying gene expression is a potential treatment in gene therapy.
Methods of Manipulation
- Transient overexpression uses vectors with constitutive promoters for high but temporary gene expression.
- Stable overexpression involves inserting a gene into the genome for long-term expression.
- RNA interference (RNAi) utilizes siRNA or shRNA to degrade target mRNA, reducing gene expression but not achieving a complete knockout.
CRISPR-Cas9 for Gene Editing
- CRISPR-Cas9 is a bacterial system adapted for genome editing.
- It utilizes Cas9 enzyme and a guide RNA (gRNA) to create double-strand breaks (DSBs) at specific genome sites.
- Gene knockout (KO) is achieved by introducing indel mutations through non-homologous end joining (NHEJ).
- Gene knock-in (KI) involves specific modifications through homologous recombination (HDR).
CRISPR Variants
- CRISPRi uses a deactivated Cas9 (dCas9) fused with repressor domains to inhibit gene expression.
- CRISPRa uses dCas9 fused to activators to activate gene transcription.
Experimental Considerations
- The choice of gene manipulation technique depends on the time frame, phenotypic outcome, and cell type used.
Challenges in Gene Manipulation
- Off-target effects can occur, requiring verification of gene manipulation using multiple approaches.
- Ethical concerns are particularly relevant for embryonic stem cell research.
- Clonal variability in stable overexpression necessitates analysis of multiple clones.
Why Study Genetic Causes of Disease?
- Understanding inheritance helps in genetic counseling and predicting disease risk.
- Identifying mutations helps in understanding disease mechanisms at a molecular level.
- It has applications in drug target identification, precision medicine, and gene therapy.
Types of Genetic Variation
- Numerical variations involve gain or loss of entire chromosomes.
- Structural variations include deletions, duplications, and inversions.
Detection Methods
- Karyotyping visualizes chromosome number and structure.
- Array CGH detects deletions or duplications by comparing test and reference DNA
- Whole genome sequencing detects both numerical and structural variations.
Types of Inheritance
- Autosomal traits are carried on non-sex chromosomes.
- Sex-linked traits are carried on the X or Y chromosomes.
- Mitochondrial traits are carried on mitochondrial DNA and are inherited maternally.
Monogenic vs.Complex Disorders
- Monogenic disorders are caused by mutations in a single gene and follow Mendelian inheritance patterns.
- Complex disorders involve multiple genes and environmental factors.
Identifying Mutations in Monogenic Disorders
- The candidate gene approach focuses on sequencing genes with a potential role in the disease.
- Whole exome/genome sequencing is unbiased and comprehensive but expensive, requiring filtering of variants based on frequency, predicted effect, and presence in healthy individuals.
Complex Traits and Genome-Wide Association Studies (GWAS)
- Complex diseases do not follow simple inheritance patterns.
- GWAS compares the DNA of affected and unaffected individuals to identify single nucleotide polymorphisms (SNPs) associated with disease.
Electron Microscopy
- This technique visualizes structures beyond the resolution of light microscopy.
- It uses electrons instead of light to provide higher resolution.
Resolution and Magnification
- Electron microscopes achieve higher resolution compared to light microscopes, enabling detailed imaging.
- Magnification does not necessarily improve image quality, and resolution is key.
Units of Measurement
- 1 millimeter (mm) = 10^ -3 meters
- 1 micrometer (µm) = 10^ -6 meters
- 1 nanometer (nm) = 10^ -9 meters
- 1 angstrom (Å) = 10^ -10 meters
Advantages of Electron Microscopy
- High-resolution imaging of sub-cellular structures
- Reveals ultrastructures like organelles, bacteria, viruses, macromolecules (proteins, nanoparticles)
- Provides structural context for biological processes
TEM vs. SEM
- Transmission electron microscopy (TEM) transmits electrons through the sample, producing 2D images of internal structures.
- Scanning electron microscopy (SEM) scans the sample with electrons, generating 3D images of surface details.
Cryo-Electron Microscopy (Cryo-EM)
- Cryo-EM helps determine protein structures at near-atomic resolution by freezing samples in a vitreous state.
- It eliminates staining artifacts, allowing for visualization of biological samples in their native state.
- It offers higher resolution than traditional EM techniques.
- It is gaining popularity due to improvements in instrumentation and data analysis.
Cryo-EM Workflow
- Protein production and purification involve recombinant methods or purification from natural sources.
- Sample freezing (plunge freezing) involves rapidly freezing samples in liquid ethane, preventing ice crystal formation.
- Data acquisition uses a specialized electron microscope with a high-resolution camera to capture images of individual protein molecules.
- Image processing involves aligning and averaging many images to improve signal-to-noise ratio and enhance resolution.
- Model building uses computational methods to fit a 3D model of the protein into the density map generated from the processed images.
Limitations of Cryo-EM
- Sample preparation can be challenging, as the sample needs to be purified and concentrated.
- Data analysis requires specialized software and expertise.
- The size of proteins that can be studied is limited due to the size of the cryo-EM grids and the ability to freeze the sample efficiently.
- There can be particle loss during freezing, especially with smaller proteins.
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Description
Test your knowledge on monoclonal antibodies, including their production process and various applications. This quiz covers hybridoma technology, clonal selection, and the advantages of using monoclonal antibodies over polyclonal ones.
