Untitled Flashcards Set

Flashcard #1
Term: What is Bisulfite Conversion?
Definition: Bisulfite conversion is a chemical treatment of DNA that selectively converts unmethylated cytosine bases to uracil, while 5-methylcytosine (methylated cytosine) remains unchanged. This differential chemical modification allows for the subsequent distinction between methylated and unmethylated cytosines through PCR amplification and DNA sequencing, making it a cornerstone technique for analyzing DNA methylation patterns.

Flashcard #2
Term: What are the common steps involved in Bisulfite Conversion?
Definition: The common steps include:

1. DNA Denaturation: Double-stranded DNA is denatured into single strands.

2. Bisulfite Treatment: Unmethylated cytosines react with sodium bisulfite, followed by desulfonation, converting them to uracil.

3. Desulfonation: Removal of sulfonate groups to finalize uracil conversion.

4. PCR Amplification: Uracil in the converted DNA template is read as thymine during PCR, while methylated cytosines are read as cytosines, allowing for the differentiation of original methylation states by subsequent sequencing.

Flashcard #3
Term: In what context is Bisulfite Conversion commonly used?
Definition: Bisulfite conversion is commonly used in epigenetics research to map DNA methylation sites across a genome, for instance, in Whole-Genome Bisulfite Sequencing (WGBS) or Reduced Representation Bisulfite Sequencing (RRBS) studies to investigate epigenetic changes associated with cancer, aging, or developmental processes.

Flashcard #4
Term: What are the key advantages of using Bisulfite Conversion?
Definition: The key advantages include:

• Single-Base Resolution: Allows for precise detection of methylation at individual CpG sites.

• Widespread Adoption: A well-established and widely used method in epigenetic research.

• Compatibility with NGS: Can be effectively combined with high-throughput sequencing for comprehensive methylation profiling.

Flashcard #5
Term: What are the major limitations or challenges of Bisulfite Conversion?
Definition: The major limitations include:

• DNA Degradation: The harsh chemical conditions of bisulfite treatment can significantly degrade DNA, requiring higher input DNA amounts.

• Incomplete Conversion/Over-conversion: Potential for incomplete conversion of unmethylated cytosines or accidental deamination of methylated cytosines, leading to inaccurate results.

• CpG Bias: Primarily focuses on CpG methylation and may not fully capture other forms of methylation (e.g., non-CpG methylation in stem cells).

Flashcard #6
Term: Why is Bisulfite Conversion considered an essential technique in epigenetics?
Definition: Bisulfite conversion is considered an essential technique in epigenetics because it provides a precise and reliable method to distinguish between methylated and unmethylated cytosines, which is crucial for understanding the intricate role of DNA methylation in gene regulation, chromatin structure, genomic imprinting, and disease development.

Flashcard #7
Term: What specific type of information does Bisulfite Conversion reveal about DNA?
Definition: Bisulfite conversion reveals the specific locations and patterns of 5-methylcytosine (5mC) modifications within a DNA sequence. This information is critical for understanding epigenetic landscapes, gene silencing, and cellular differentiation.

Flashcard #8
Term: What is DNA Methylation?
Definition: DNA methylation is a crucial epigenetic modification involving the addition of a methyl group (usually CH33​) to a DNA base, most commonly at the 5-carbon position of a cytosine residue within CpG dinucleotides. This modification does not alter the DNA sequence itself but plays a significant role in regulating gene expression, silencing transposable elements, maintaining chromatin structure, and influencing various biological processes from embryonic development to disease progression.

Flashcard #9
Term: What is PCR Amplification?
Definition: PCR amplification is a molecular biology technique used to generate millions of copies of a specific DNA segment from a very small initial sample. It is an enzymatic process that rapidly amplifies target DNA sequences by cycling through denaturation, annealing, and extension steps, driven by a DNA polymerase enzyme.

Flashcard #10
Term: What are the common steps involved in PCR Amplification?
Definition: The common steps involved in PCR amplification, iterated over 20-40 cycles, are:

1. Denaturation: The DNA template is heated (typically 94−98extoextC94−98extoextC) to separate its double strands into single strands.

2. Annealing: The reaction is cooled (typically 50−65extoextC50−65extoextC) to allow short, synthetic DNA primers to bind (anneal) to complementary sequences on the single-stranded DNA templates.

3. Extension (Elongation): The temperature is raised (typically 70−75extoextC70−75extoextC) to activate a heat-stable DNA polymerase (e.g., Taq polymerase) which synthesizes new DNA strands by extending the primers, complementary to the template.

Flashcard #11
Term: In what context is PCR Amplification commonly used?
Definition: PCR amplification is widely used in forensic science to amplify minute DNA samples from crime scenes for DNA fingerprinting, in medical diagnostics for detecting viral or bacterial pathogens (e.g., SARS-CoV-2), and in genetics research for cloning genes or preparing DNA for sequencing.

Flashcard #12
Term: What are the key advantages of using PCR Amplification?
Definition: The key advantages include:

• High Sensitivity: Can amplify DNA from very small starting amounts.

• Specificity: Primers ensure amplification of a specific target sequence.

• Speed and Efficiency: Rapidly produces large quantities of DNA in a relatively short time.

• Versatility: Adaptable for numerous applications beyond simple amplification, such as mutation detection, gene expression analysis, and genotyping.

Flashcard #13
Term: What are the major limitations or challenges of PCR Amplification?
Definition: The major limitations include:

• Contamination Risk: Highly sensitive, making it prone to contamination from exogenous DNA, leading to false positives.

• Primer Design Sensitivity: Poorly designed primers can lead to non-specific amplification or primer dimers.

• Limited Amplicon Size: Generally less efficient for amplifying very long DNA fragments (typically best for sequences up to a few kilobases).

Flashcard #14
Term: Why is PCR Amplification considered an essential technique?
Definition: PCR amplification is an indispensable technique because it enables the study and manipulation of specific DNA sequences from limited samples, revolutionizing fields like molecular diagnostics, genetic research, forensics, and evolutionary biology by making DNA analysis accessible and highly efficient.

Flashcard #15
Term: What specific type of information does PCR Amplification reveal about DNA?
Definition: PCR amplification reveals the presence or absence of a specific DNA sequence, its quantity (when combined with qPCR), and can also be used as a preparatory step for further analysis, such as sequencing or cloning, to determine the exact sequence or manipulate the DNA.

Flashcard #16
Term: What is Next Generation Sequencing (NGS)?
Definition: Next-Generation Sequencing (NGS), also known as massively parallel sequencing, refers to a suite of high-throughput DNA sequencing technologies that have revolutionized genomic research. Unlike traditional Sanger sequencing, NGS allows for the simultaneous sequencing of millions of DNA fragments, generating vast amounts of sequence data rapidly and at a significantly lower cost per base.

Flashcard #17
Term: What are the common steps involved in Next Generation Sequencing (NGS)?
Definition: While platforms vary (e.g., Illumina, Oxford Nanopore), general steps include:

1. Library Preparation: Genomic DNA/RNA is fragmented, adaptors are ligated to the ends, and fragments are often amplified to create a sequencing library.

2. Cluster Generation/Template Preparation: Library fragments are immobilized on a solid surface (e.g., flow cell) and amplified to create clonal clusters of identical DNA templates.

3. Sequencing by Synthesis/Ligation/Ion Sensing: Nucleotides are incorporated one by one with fluorescent labels (or other detection methods), and images are captured after each cycle to identify the incorporated base. Alternatively, ions released during nucleotide incorporation are detected.

4. Data Analysis: Raw sequence reads are aligned to a reference genome or assembled de novo, followed by variant calling, gene expression quantification, or other downstream bioinformatics analyses.

Flashcard #18
Term: In what context is Next Generation Sequencing (NGS) commonly used?
Definition: NGS is extensively used for whole-genome sequencing to identify genetic variations, for RNA sequencing (RNA-seq) to quantify gene expression, for ChIP-seq to map protein-DNA interactions, and for clinical diagnostics to identify disease-causing mutations or characterize microbial communities in metagenomics studies.

Flashcard #19
Term: What are the key advantages of using Next Generation Sequencing (NGS)?
Definition: The key advantages include:

• High Throughput: Generates billions of bases of sequence data in a single run.

• Cost-Effectiveness: Significantly reduces the cost per base compared to Sanger sequencing for large-scale projects.

• Speed: Much faster turnaround times for comprehensive genomic analysis.

• Versatility: Applicable to a wide range of genomic and epigenomic studies, including DNA, RNA, and protein-DNA interactions.

Flashcard #20
Term: What are the major limitations or challenges of Next Generation Sequencing (NGS)?
Definition: The major limitations include:

• Computational Demands: Generates massive datasets requiring significant computational power, storage, and specialized bioinformatics expertise for analysis.

• Short Read Lengths (for some platforms): Many common NGS platforms produce relatively short reads, which can make de novo assembly or sequencing through repetitive regions challenging.

• Complexity: Library preparation and data analysis workflows can be complex and require careful optimization.

Flashcard #21
Term: Why is Next Generation Sequencing (NGS) considered an essential technique?
Definition: NGS is considered an essential technique because it has enabled unprecedented insights into genomics, transcriptomics, and epigenomics by providing the capability to rapidly and affordably sequence entire genomes, identify subtle genetic variations, quantify gene expression, and explore complex molecular interactions at a massive scale, driving advancements in basic research, diagnostics, and personalized medicine.

Flashcard #22
Term: What specific type of information does Next Generation Sequencing (NGS) reveal about DNA and RNA?
Definition: NGS reveals comprehensive genomic information, including DNA sequence variations (SNPs, indels, structural variants), gene expression levels (from RNA sequencing), epigenetic modifications (from bisulfite or ChIP-sequencing), and microbial community composition (from metagenomics). It provides a holistic view of the genetic and molecular landscape.

Flashcard #23
Term: What is qPCR (quantitative Polymerase Chain Reaction)?
Definition: qPCR (quantitative Polymerase Chain Reaction), also known as real-time PCR, is a laboratory technique based on PCR that monitors the amplification of a targeted DNA molecule during the PCR process, in real time. It quantifies the amount of DNA or RNA (after reverse transcription) present in a sample by measuring the fluorescence intensity emitted by a reporter molecule that increases proportionally to the amount of amplified product.

Flashcard #24
Term: What are the common steps involved in qPCR (quantitative Polymerase Chain Reaction)?
Definition: The common steps involved in qPCR include:

1. Reverse Transcription (for RNA): If starting with RNA, it's first converted to cDNA using reverse transcriptase.

2. Reaction Setup: Master mix (containing DNA polymerase, dNTPs, buffer, primers, and a fluorescent reporter dye/probe) is combined with the DNA/cDNA template.

3. Thermocycling: The reaction undergoes repeated cycles of denaturation, annealing, and extension, similar to conventional PCR.

4. Fluorescence Detection: A real-time PCR instrument measures the accumulation of fluorescence signal at each cycle as the amplification progresses. The cycle threshold (Ct) value, which is the cycle number at which fluorescence crosses a threshold, is inversely proportional to the initial amount of target nucleic acid.

Flashcard #25
Term: In what context is qPCR (quantitative Polymerase Chain Reaction) commonly used?
Definition: qPCR is widely used in medical diagnostics for rapid detection and quantification of viral loads (e.g., HIV, Hepatitis C) or bacterial pathogens, in gene expression studies to measure mRNA levels in response to treatments or disease conditions, and for evaluating genetically modified organisms (GMOs).

Flashcard #26
Term: What are the key advantages of using qPCR (quantitative Polymerase Chain Reaction)?
Definition: The key advantages include:

• Quantification: Provides absolute or relative quantification of target nucleic acids.

• Speed: Faster than conventional PCR for analyzing multiple samples because it avoids post-PCR gel electrophoresis.

• High Sensitivity and Specificity: Can detect very low quantities of target molecules with high specificity due to primer and probe design.

• Reduced Contamination Risk: Closed-tube system minimizes post-PCR handling, reducing contamination risk.

Flashcard #27
Term: What are the major limitations or challenges of qPCR (quantitative Polymerase Chain Reaction)?
Definition: The major limitations include:

• Inhibitors: Substances in the sample can inhibit the PCR reaction, leading to inaccurate quantification.

• Standard Curve Dependency (for absolute quantification): Requires a reliable standard curve generated from known concentrations of target DNA/RNA.

• Primer/Probe Design: Critical for accurate and specific results; poor design can lead to non-specific amplification.

• Cost: Instruments and specialized reagents can be more expensive than conventional PCR setups.

Flashcard #28
Term: Why is qPCR (quantitative Polymerase Chain Reaction) considered an essential technique?
Definition: qPCR is considered an essential technique because it offers real-time, quantitative, and highly sensitive detection and measurement of specific nucleic acid targets. This capability is critical for applications demanding precise quantification, such as viral load monitoring, gene expression profiling, and detection of rare genetic events, significantly impacting clinical diagnostics and molecular research.

Flashcard #29
Term: What specific type of information does qPCR (quantitative Polymerase Chain Reaction) reveal about nucleic acids?
Definition: qPCR reveals the initial quantity (absolute or relative) of a specific DNA or RNA sequence in a sample. It provides a measure of gene expression levels (mRNA), pathogen load, or the number of specific genetic targets. It also indicates the presence or absence of a target sequence.

Flashcard #30
Term: What is CRISPR/Cas9?
Definition: CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) is a revolutionary gene editing technology that allows scientists to make precise, targeted modifications to DNA within living cells and organisms. It uses a guide RNA (gRNA) molecule to direct the Cas9 nuclease enzyme to a specific genomic location, where it creates a double-strand break in the DNA, which cells then repair via natural repair mechanisms (NHEJ or HDR), leading to gene knockout, insertion, or correction.

Flashcard #31
Term: What are the common steps involved in CRISPR/Cas9 gene editing?
Definition: The common steps involved in CRISPR/Cas9 gene editing include:

1. gRNA Design: A guide RNA (gRNA) is designed to be complementary to the specific DNA sequence targeted for modification.

2. Delivery: The Cas9 nuclease (or its encoding gene) and the gRNA are delivered into the target cells (e.g., via plasmids, viral vectors, or ribonucleoprotein).

3. Targeting and Cleavage: The gRNA directs Cas9 to the complementary DNA sequence, where Cas9 creates a double-strand break.

4. DNA Repair: The cell's natural DNA repair mechanisms activate: Non-Homologous End Joining (NHEJ) often leads to gene knockout by introducing indels, while Homology-Directed Repair (HDR), if a repair template is provided, allows for precise gene insertion or correction.

Flashcard #32
Term: In what context is CRISPR/Cas9 commonly used?
Definition: CRISPR/Cas9 is being widely explored for therapeutics to correct disease-causing mutations (e.g., in sickle cell anemia or cystic fibrosis), for creating genetically modified organisms (GMOs) with improved traits in agriculture, and for basic research to understand gene function by creating gene knockouts in cell lines or animal models.

Flashcard #33
Term: What are the key advantages of using CRISPR/Cas9?
Definition: The key advantages include:

• Precision and Specificity: Highly accurate in targeting specific DNA sequences.

• Simplicity and Ease of Use: Relatively simpler and easier to design and execute compared to older gene editing technologies.

• Versatility: Can be used for gene knockout, gene insertion, gene correction, and even gene activation/repression (CRISPRa/i).

• Cost-Effectiveness: More affordable than previous methods, making it widely accessible.

Flashcard #34
Term: What are the major limitations or challenges of CRISPR/Cas9?
Definition: The major limitations include:

• Off-Target Effects: Potential for Cas9 to cleave DNA at unintended sites, leading to unwanted mutations.

• Delivery Challenges: Efficient and safe delivery of CRISPR components into specific cell types or tissues in vivo remains a significant hurdle for therapeutic applications.

• Ethical Concerns: Raises ethical considerations, particularly regarding germline editing and its potential implications for human heredity.

Flashcard #35
Term: Why is CRISPR/Cas9 considered an essential technology?
Definition: CRISPR/Cas9 is considered an essential technology because it provides an unparalleled tool for precise and efficient genome engineering, opening up vast possibilities for basic biological research, the development of new gene therapies, and advancements in biotechnology and agriculture. Its ability to directly modify the genetic code has transformed our approach to understanding and treating genetic diseases.

Flashcard #36
Term: What specific type of information does CRISPR/Cas9 reveal or allow us to obtain?
Definition: CRISPR/Cas9 allows for the modification of the genetic sequence at specific locations. It reveals the functional consequences of altering specific genes, such as the impact of gene knockout on protein function or cellular phenotype, or the successful correction of a mutation. It provides direct evidence of gene function and regulatory mechanisms.

Flashcard #37
Term: What is siRNA (Small Interfering RNA)?
Definition: siRNA (small interfering RNA) are short (typically 20-25 nucleotides long), double-stranded RNA molecules that play a crucial role in RNA interference (RNAi), a biological process by which gene expression is silenced at the post-transcriptional level. siRNA molecules guide the RNA-induced silencing complex (RISC) to complementary messenger RNA (mRNA) transcripts, leading to their degradation and, consequently, a reduction in the production of the corresponding protein.

Flashcard #38
Term: What are the common steps involved in siRNA-mediated gene silencing?
Definition: The common steps involved in siRNA-mediated gene silencing include:

1. Delivery: Synthetic siRNA molecules are introduced into cells.

2. RISC Loading: The siRNA is incorporated into the RNA-induced silencing complex (RISC), where one strand (the passenger strand) is cleaved and discarded, leaving the guide strand.

3. Target Recognition: The guide strand directs RISC to a complementary mRNA transcript.

4. mRNA Cleavage: Caspase-dependent ribonucleases within RISC cleave the target mRNA, leading to its degradation.

5. Gene Silencing: The degradation of mRNA prevents its translation into protein, effectively silencing the gene.

Flashcard #39
Term: In what context is siRNA (Small Interfering RNA) commonly used?
Definition: siRNA is frequently used in research to study gene function by knocking down the expression of specific genes and observing the resulting phenotypic changes. It is also being investigated as a therapeutic agent for various diseases, such as viral infections (e.g., RSV) and certain cancers, by targeting disease-associated genes.

Flashcard #40
Term: What are the key advantages of using siRNA (Small Interfering RNA)?
Definition: The key advantages include:

• Specificity: Highly specific in targeting and silencing a particular gene through sequence complementarity.

• Broad Applicability: Can be used to silence virtually any gene for which sequence information is available.

• Reversibility: Gene silencing is transient, making it useful for studying gene function without permanent genomic alteration.

Flashcard #41
Term: What are the major limitations or challenges of siRNA (Small Interfering RNA)?
Definition: The major limitations include:

• Off-Target Effects: siRNA can sometimes bind to and silence unintended genes due to partial sequence complementarity, leading to confounding results.

• Delivery Challenges: Efficient and stable delivery of siRNA into target cells or tissues in vivo remains a significant hurdle for therapeutic applications.

• Transient Effect: The silencing effect is temporary, requiring repeated administration for sustained knockdown.

• Immunostimulation: High concentrations or certain sequences of siRNA can trigger an innate immune response.

Flashcard #42
Term: Why is siRNA (Small Interfering RNA) considered an essential tool?
Definition: siRNA is considered an essential tool because it provides a powerful and specific method for transiently reducing the expression of a targeted gene. This enables researchers to investigate gene function by observing the consequences of its absence, and it holds therapeutic potential by silencing genes that contribute to disease, offering a gene-specific approach to drug discovery and treatment.

Flashcard #43
Term: What specific type of information does siRNA (Small Interfering RNA) reveal?
Definition: siRNA allows researchers to infer the function of a specific gene by observing the phenotypic consequences of its reduced expression. It directly shows that a particular gene's protein product is necessary for a certain cellular process or contributes to a disease state when its expression is lowered.

Flashcard #44
Term: What is Chromatin Immunoprecipitation (ChIP)?
Definition: Chromatin Immunoprecipitation (ChIP) is a powerful molecular biology technique used to investigate the interactions between proteins and DNA in vivo. It allows for the identification of specific genomic regions to which a particular protein (such as transcription factors, histone modifications, or chromatin remodeling enzymes) is bound within the context of living cells, providing insights into gene regulation and chromatin structure.

Flashcard #45
Term: What are the common steps involved in Chromatin Immunoprecipitation (ChIP)?
Definition: The common steps involved in ChIP include:

1. Cross-linking: Cells are treated with formaldehyde to covalently cross-link proteins to DNA, preserving protein-DNA interactions.

2. Chromatin Fragmentation: The cross-linked chromatin is fragmented into small, manageable pieces (e.g., by sonication or enzymatic digestion).

3. Immunoprecipitation: An antibody specific to the protein of interest is used to selectively "pull down" (immunoprecipitate) the protein-DNA complexes.

4. Washing and Reversal of Cross-links: Non-specific binding is removed by washes, and the cross-links are reversed to release the DNA fragments.

5. DNA Purification and Analysis: The purified DNA fragments are then analyzed using techniques like qPCR (ChIP-qPCR) or Next-Generation Sequencing (ChIP-seq) to identify the specific DNA regions enriched by the protein binding.

Flashcard #46
Term: In what context is Chromatin Immunoprecipitation (ChIP) commonly used?
Definition: ChIP is widely used to identify the binding sites of transcription factors across the genome, to map specific histone modifications associated with active or repressed gene expression, and to understand how chromatin structure influences gene regulation in various biological processes, such as development, disease, and cellular differentiation.

Flashcard #47
Term: What are the key advantages of using Chromatin Immunoprecipitation (ChIP)?
Definition: The key advantages include:

• Genome-wide Mapping: When combined with sequencing (ChIP-seq), it can provide a high-resolution, global map of protein-DNA interactions.

• Direct Interaction: Directly identifies DNA regions bound by specific proteins in vivo.

• Versatility: Applicable to various DNA-binding proteins, including transcription factors, histones, and polymerases.

Flashcard #48
Term: What are the major limitations or challenges of Chromatin Immunoprecipitation (ChIP)?
Definition: The major limitations include:

• Antibody Quality: Requires high-quality, specific antibodies; non-specific antibodies can lead to false positives.

• Input Material: Requires a substantial amount of starting material (cells/tissue).

• Cross-linking Optimization: Over- or under-cross-linking can affect efficiency and resolution.

• Computational Complexity: ChIP-seq data analysis is computationally intensive and requires bioinformatics expertise.

Flashcard #49
Term: Why is Chromatin Immunoprecipitation (ChIP) considered an essential technique?
Definition: ChIP is considered an essential technique because it provides direct evidence of protein-DNA interactions within the native chromatin context of a cell. This allows researchers to pinpoint regulatory elements, understand the mechanisms of gene expression control, and unlock insights into how epigenetic modifications and transcription factors orchestrate cellular processes.

Flashcard #50
Term: What specific type of information does Chromatin Immunoprecipitation (ChIP) reveal about protein-DNA interactions?
Definition: ChIP identifies the specific genomic regions (e.g., promoters, enhancers) where a particular protein binds in vivo. It reveals the targets of transcription factors, the locations of specific histone modifications (indicating active or repressed chromatin), and generally helps to map the regulatory landscape of the genome, providing insights into gene regulation and chromatin dynamics.

Flashcard #51
Term: What is Whole Genome Sequencing (WGS)?
Definition: Whole Genome Sequencing (WGS) is a comprehensive laboratory process that determines the complete DNA sequence of an organism's entire genome at a single time. Unlike targeted sequencing approaches, WGS captures all the genetic information (coding and non-coding regions) present in an individual's DNA, providing a foundational dataset for understanding genetic variation, predisposition to disease, and evolutionary relationships.

Flashcard #52
Term: What are the common steps involved in Whole Genome Sequencing (WGS)?
Definition: The common steps involved in WGS using NGS platforms include:

1. DNA Library Preparation: Genomic DNA is extracted, fragmented into smaller pieces, and then size-selected. Adapter sequences are ligated to both ends of these fragments to facilitate binding to the sequencing platform.

2. Clonal Amplification/Cluster Generation: The prepared library fragments are amplified to create millions of identical copies (clusters) on a flow cell or bead, which are then ready for sequencing.

3. Sequencing: Clonal clusters are sequenced simultaneously using various NGS technologies (e.g., sequencing by synthesis, ion semiconductor sequencing), generating millions of short reads.

4. Data Analysis: Raw sequence reads are aligned to a reference genome. Subsequent bioinformatics analyses are performed for variant calling (identifying SNPs, indels, structural variants), copy number variation analysis, and functional annotation.

Flashcard #53
Term: In what context is Whole Genome Sequencing (WGS) commonly used?
Definition: WGS is used in clinical diagnostics to identify rare disease-causing mutations, in oncology to characterize tumor genomics and guide personalized cancer treatment, in public health to track pathogen outbreaks, and in population genetics and evolutionary biology to study human diversity and evolutionary history.

Flashcard #54
Term: What are the key advantages of using Whole Genome Sequencing (WGS)?
Definition: The key advantages include:

• Comprehensive Information: Captures virtually all genetic variations (SNPs, indels, CNVs, structural variants) across the entire genome, including non-coding regions.

• Discovery Power: Enables the discovery of novel genetic variants or disease-associated loci that might be missed by targeted sequencing.

• Single Test: Provides maximum genetic information from a single experiment.

• Future-Proofing: The complete genome sequence can be re-analyzed later as new knowledge emerges without needing to re-sequence.

Flashcard #55
Term: What are the major limitations or challenges of Whole Genome Sequencing (WGS)?
Definition: The major limitations include:

• High Cost: Despite decreasing costs, it remains more expensive than targeted sequencing for single genes or exons.

• Computational Demands: Generates massive datasets that require significant computational resources, storage, and advanced bioinformatics expertise for analysis.

• Interpretation Complexity: Interpreting the functional significance of non-coding variants can be challenging.

• Ethical Concerns: Raises privacy and ethical considerations due to the vast amount of individual genetic information generated.

Flashcard #56
Term: Why is Whole Genome Sequencing (WGS) considered an essential technique?
Definition: WGS is considered an essential technique because it offers the most comprehensive view of an organism's genetic makeup, enabling unparalleled insights into basic biology, disease mechanisms, and evolution. It has become a cornerstone for precise diagnostics, therapeutic target identification, and personalized medicine by providing a complete genetic blueprint.

Flashcard #57
Term: What specific type of information does Whole Genome Sequencing (WGS) reveal?
Definition: WGS reveals the entire DNA sequence of an organism's genome, including all coding and non-coding regions. It identifies all types of sequence variations present in an individual, providing the complete genetic blueprint and serving as a critical resource for understanding genetic predispositions, disease etiology, and evolutionary relationships.

Flashcard #58
Term: What is Digital PCR (dPCR)?
Definition: Digital PCR (dPCR) is an advanced nucleic acid quantification technique that provides absolute quantification of target DNA or RNA molecules by partitioning a sample into thousands to millions of individual, miniature reactions. After amplification, the number of positive (containing target) and negative (no target) partitions is counted, allowing for a precise, absolute digital readout of the initial target concentration, without the need for a standard curve.

Flashcard #59
Term: What are the common steps involved in Digital PCR (dPCR)?
Definition: The general steps involved in dPCR include:

1. Sample Partitioning: The sample containing the DNA/RNA target, along with PCR reagents, is partitioned into a large number of discrete, isolated reaction volumes (e.g., droplets in an emulsion, or wells on a microfluidic chip).

2. PCR Amplification: Each individual partition undergoes PCR amplification until the end point. Partitions initially containing at least one target molecule will become fluorescent (positive), while those with no target remain non-fluorescent (negative).

3. Digital Readout: An instrument counts the number of positive and negative partitions. Statistical analysis (Poisson distribution) is then used to calculate the absolute concentration of the target nucleic acid in the original sample.

Flashcard #60
Term: In what context is Digital PCR (dPCR) commonly used?
Definition: dPCR is used for highly sensitive liquid biopsy assays to detect rare cancer mutations in circulating tumor DNA (ctDNA), for precise quantification of viral load in infectious diseases (e.g., HIV, HBV), for absolute quantification of gene expression, for validating NGS variants, and for quality control of gene therapy products.

Flashcard #61
Term: What are the key advantages of using Digital PCR (dPCR)?
Definition: The key advantages include:

• Absolute Quantification: Provides direct, absolute counts of target molecules without a standard curve.

• High Precision and Reproducibility: Less sensitive to inhibitors and variations in reaction efficiency compared to qPCR.

• High Sensitivity: Exceptional sensitivity for detecting and quantifying rare targets or subtle differences in concentration.

• Robustness: More resistant to PCR inhibitors present in complex samples.

Flashcard #62
Term: What are the major limitations or challenges of Digital PCR (dPCR)?
Definition: The major limitations include:

• Lower Throughput: Currently, dPCR has lower sample throughput compared to qPCR for large studies.

• Higher Cost per Sample: Reagents and consumables can be more expensive than qPCR.

• Limited Multiplexing: While improving, multiplexing (detecting multiple targets simultaneously) is generally more challenging than with qPCR.

• Instrument Specificity: Requires specialized dPCR instruments for partitioning and readout.

Flashcard #63
Term: Why is Digital PCR (dPCR) considered an essential technique?
Definition: dPCR is considered an essential technique because it offers unparalleled precision and sensitivity for the absolute quantification of nucleic acids. Its ability to detect and quantify rare target molecules with high accuracy makes it indispensable for applications where even subtle changes in concentration are critical, such as liquid biopsies for cancer detection or quantifying gene editing events.

Flashcard #64
Term: What specific type of information does Digital PCR (dPCR) reveal about nucleic acids?
Definition: dPCR reveals the precise, absolute concentration (number of copies per unit volume) of a specific DNA or RNA target in a sample. It tells you exactly how many molecules of your target were initially present, making it invaluable for detecting low-abundance targets, quantifying viral loads, or validating cell line genome edits.

Flashcard #65
Term: What is Sanger Sequencing?
Definition: Sanger sequencing, also known as the chain-termination method or dideoxy sequencing, is a first-generation DNA sequencing technique developed by Frederick Sanger. It determines the nucleotide sequence of a DNA fragment by selectively incorporating dideoxynucleotides (ddNTPs), which lack a 3'-hydroxyl group, into a growing DNA strand, thereby terminating its synthesis at specific bases. This generates a series of DNA fragments of varying lengths, which are then separated and detected to infer the sequence.

Flashcard #66
Term: What are the common steps involved in Sanger Sequencing?
Definition: The common steps involved in automated Sanger sequencing include:

1. Reaction Setup: A sequencing reaction mix is prepared, containing the DNA template, a primer, DNA polymerase, standard deoxynucleotides (dNTPs), and a small amount of four different fluorescently labeled dideoxynucleotides (ddNTPs) (A, T, C, G).

2. Chain Termination: During DNA synthesis, controlled incorporation of ddNTPs randomly terminates DNA strand elongation at every position where that specific ddNTP is incorporated, creating a ladder of fluorescently labeled fragments of varying lengths.

3. Capillary Electrophoresis: The fragmented DNA products are separated by size using high-resolution capillary electrophoresis. As fragments pass a laser detector, the fluorescent label of the terminal ddNTP is excited, and its emission wavelength is recorded.

4. Sequence Generation: A computer software converts the detected fluorescence signals into a chromatogram, representing the DNA sequence.

Flashcard #67
Term: In what context is Sanger Sequencing commonly used?
Definition: Sanger sequencing is still widely used for sequencing individual genes or small DNA fragments, for confirming variants identified by NGS (gold standard validation), for microbial identification (e.g., 16S rRNA gene sequencing), and for re-sequencing specific regions in clinical diagnostics where high accuracy for a single target is required.

Flashcard #68
Term: What are the key advantages of using Sanger Sequencing?
Definition: The key advantages include:

• High Accuracy: Considered the "gold standard" for sequence accuracy over short-to-medium read lengths (500−1000extbp500−1000extbp).

• Long Read Lengths: Can typically produce longer contiguous reads compared to many NGS platforms, useful for resolving repetitive regions difficult for short reads.

• Simplicity and Reliability: Relatively straightforward workflow and robust for single-gene sequencing.

• Cost-Effective for Small Projects: More economical than NGS for sequencing a few samples or specific targets.

Flashcard #69
Term: What are the major limitations or challenges of Sanger Sequencing?
Definition: The major limitations include:

• Low Throughput: Capable of sequencing only one DNA fragment at a time, making it impractical for large-scale genomic projects.

• High Cost per Base (for large projects): Becomes economically inefficient for whole-genome or exome sequencing.

• Requires Purified DNA: Sensitive to impure DNA samples.

• Difficulty with Complex Samples: Challenging for sequencing highly repetitive regions or mixed samples.

Flashcard #70
Term: Why is Sanger Sequencing considered an essential technique?
Definition: Sanger sequencing, despite the advent of NGS, is considered an essential technique because it offers unmatched accuracy for validating novel variants identified by high-throughput methods and remains the benchmark for sequencing individual genes or specific genomic regions where high confidence and longer read lengths are critical, especially in clinical diagnostics and quality control.

Flashcard #71
Term: What specific type of information does Sanger Sequencing reveal about DNA?
Definition: Sanger sequencing reveals the precise nucleotide sequence (A, T, C, G) of a specific, relatively short DNA fragment. It can identify single nucleotide polymorphisms (SNPs), small insertions and deletions (indels), and confirm the exact sequence of a cloned gene or a region of interest.

Flashcard #72
Term: What is LAMP (Loop-mediated Isothermal Amplification)?
Definition: LAMP (Loop-mediated Isothermal Amplification) is a rapid, highly sensitive, and cost-effective nucleic acid amplification technique. Unlike PCR, LAMP is performed at a single, constant temperature (isothermal), eliminating the need for a thermal cycler. It uses a unique set of 4 to 6 primers that specifically recognize 6 to 8 distinct regions on the target DNA, leading to a highly efficient strand displacement DNA synthesis and rapid accumulation of a large amount of DNA.

Flashcard #73
Term: What are the common steps involved in LAMP (Loop-mediated Isothermal Amplification)?
Definition: The common steps involved in LAMP include:

1. Reaction Setup: A reaction mix is prepared containing the DNA/RNA template, Bst DNA polymerase (which has strand displacement activity), and a unique set of 4-6 primers (2 outer, 2 inner, and sometimes 2 loop primers).

2. Isothermal Incubation: The reaction is incubated at a constant temperature (typically 60−65extoextC60−65extoextC) for typically 15−6015−60 minutes.

3. Self-Priming and Amplification: The inner primers initiate DNA synthesis, and the outer primers displace the newly synthesized strands. The inner primers then self-anneal to form loop structures at both ends of the amplicon, allowing continuous and rapid amplification in a highly efficient auto-cycling manner.

4. Detection: Amplification can be detected in various ways, often visually, (e.g., turbidity due to magnesium pyrophosphate precipitates, color change from pH indicators, or fluorescence from intercalating dyes).

Flashcard #74
Term: In what context is LAMP (Loop-mediated Isothermal Amplification) commonly used?
Definition: LAMP is widely used for point-of-care diagnostics for infectious diseases (e.g., COVID-19, malaria, tuberculosis) due to its simplicity and speed, for food safety testing to detect pathogens or adulterants, and for environmental monitoring to identify specific microbes in water or soil samples.

Flashcard #75
Term: What are the key advantages of using LAMP (Loop-mediated Isothermal Amplification)?
Definition: The key advantages include:

• Isothermal: Does not require a thermal cycler, simplifying equipment needs, making it suitable for field or low-resource settings.

• Rapid: Generates a large amount of DNA very quickly, often within 15-60 minutes.

• High Sensitivity and Specificity: Uses multiple primers recognizing many target regions, providing high detection limits and specificity.

• Visual Detection: Many detection methods are simple and visual, requiring minimal specialized equipment.

Flashcard #76
Term: What are the major limitations or challenges of LAMP (Loop-mediated Isothermal Amplification)?
Definition: The major limitations include:

• Primer Design Complexity: Designing the 4-6 specific LAMP primers is more complex than PCR primer design and requires specialized software.

• Product Accumulation: Produces massive amounts of DNA, which can easily lead to carry-over contamination if not handled carefully.

• No Quantitative Data (typically): Primarily a qualitative (presence/absence) method; accurate quantification can be challenging.

• Multiplexing Challenges: Multiplexing multiple targets in a single reaction is difficult.

Flashcard #77
Term: Why is LAMP (Loop-mediated Isothermal Amplification) considered an essential technique?
Definition: LAMP is considered an essential technique because it offers a rapid, highly sensitive, and robust method for nucleic acid amplification under isothermal conditions, making it perfectly suited for accessible, on-site diagnostics, especially in resource-limited settings. Its simplicity and speed have made it invaluable for rapid pathogen detection and field-based molecular testing.

Flashcard #78
Term: What specific type of information does LAMP (Loop-mediated Isothermal Amplification) reveal?
Definition: LAMP reveals the presence or absence of a specific DNA or RNA target sequence in a sample. It provides a highly sensitive and rapid qualitative detection method for pathogens, specific genes, or genetic markers, indicating contamination, infection, or the presence of a target of interest.

Flashcard #79
Term: What is ATAC-seq?
Definition: ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) is a rapidly growing molecular biology technique used to investigate genome-wide chromatin accessibility. It identifies regions of open, active chromatin by using a hyperactive transposase enzyme (Tn5) loaded with sequencing adaptors. This enzyme preferentially cuts and tags accessible DNA regions, allowing researchers to map nucleosome-free regions and thus indirectly identify regulatory elements like promoters and enhancers that are actively involved in gene expression.

Flashcard #80
Term: What are the common steps involved in ATAC-seq?
Definition: The common steps involved in ATAC-seq include:

1. Cell Lysis and Nuclei Isolation: Cells are gently lysed to obtain intact nuclei.

2. Tagmentation: The Tn5 transposase, pre-loaded with sequencing adapters, simultaneously fragments and ligates (tags) adapters into accessible (open) chromatin regions within the intact nuclei. Inaccessible regions, bound by nucleosomes or other proteins, are protected from Tn5 insertion.

3. PCR Amplification: The tagged DNA fragments are PCR amplified to add complete sequencing adapters and unique indexing primers.

4. Sequencing and Data Analysis: The amplified library is sequenced using NGS. Reads are mapped to a reference genome, and areas with high read density correspond to regions of accessible chromatin.

Flashcard #81
Term: In what context is ATAC-seq commonly used?
Definition: ATAC-seq is widely used in epigenomics and gene regulation studies to identify active regulatory elements (promoters, enhancers) in different cell types or disease states, to understand how chromatin accessibility changes during cellular differentiation or in response to external stimuli, and to link transcription factor binding with gene expression.

Flashcard #82
Term: What are the key advantages of using ATAC-seq?
Definition: The key advantages include:

• High Resolution: Provides single-nucleosome resolution of chromatin accessibility.

• Low Cell Input: Can be performed with relatively few cells (down to hundreds or even single cells for scATAC-seq).

• Speed and Simplicity: Quicker and simpler library preparation compared to older methods like DNase-seq.

• Broad Applicability: Useful for identifying active regulatory regions indicative of gene expression.

Flashcard #83
Term: What are the major limitations or challenges of ATAC-seq?
Definition: The major limitations include:

• Requires Intact Nuclei: Sample preparation requires careful handling to preserve nuclear integrity.

• Susceptibility to High Mitochondrial DNA: High mitochondrial DNA content can lead to a large proportion of non-informative reads, necessitating purification or careful analysis.

• Computational Demands: Data analysis is computationally intensive, requiring specialized bioinformatics tools and expertise to interpret peaks and relate them to gene regulation.

• Coverage Requirements: Deeper sequencing is often needed for identifying smaller or less abundant peaks.

Flashcard #84
Term: Why is ATAC-seq considered an essential technique?
Definition: ATAC-seq is considered an essential technique because it offers a rapid, sensitive, and genome-wide method for mapping chromatin accessibility, a key indicator of transcriptional activity. By identifying open chromatin regions, ATAC-seq provides crucial insights into the regulatory landscape of the genome, revealing active promoters, enhancers, and transcription factor binding sites, which is fundamental to understanding gene regulation and cell identity.

Flashcard #85
Term: What specific type of information does ATAC-seq reveal about chromatin?
Definition: ATAC-seq reveals regions of open, accessible chromatin across the genome. This indicates genomic loci that are not tightly packed by nucleosomes and are potentially available for transcription factor binding and gene expression. It provides insights into active regulatory elements (promoters, enhancers) and the overall transcriptional potential of a cell or tissue.

Flashcard #86
Term: What is Immunofluorescence (IF)?
Definition: Immunofluorescence (IF) is a versatile microscopy technique that utilizes the specificity of antibodies to visualize and localize specific proteins or antigens within cells or tissue sections. Fluorescent dyes are chemically linked to antibodies, which then bind to their target antigens, allowing their spatial distribution, abundance, and interactions to be observed under a fluorescence microscope.

Flashcard #87
Term: What are the common steps involved in Immunofluorescence (IF)?
Definition: The common steps involved in immunofluorescence include:

1. Sample Preparation: Cells or tissue sections are fixed (to preserve cellular structures and antigens) and permeabilized (to allow antibodies to enter).

2. Blocking: Samples are incubated with a blocking solution to prevent non-specific binding of antibodies.

3. Primary Antibody Incubation: The sample is incubated with a primary antibody (unlabeled) that specifically binds to the target protein.

4. Washing: Excess unbound primary antibody is washed away.

5. Secondary Antibody Incubation (Indirect IF): For indirect IF, a fluorescently labeled secondary antibody (which binds to the primary antibody) is added. In direct IF, the primary antibody itself is fluorescently labeled, and this step is skipped.

6. Washing: Excess unbound secondary antibody is washed away.

7. Mounting and Imaging: The sample is mounted with an anti-fade mounting medium and visualized using a fluorescence microscope.

Flashcard #88
Term: In what context is Immunofluorescence (IF) commonly used?
Definition: IF is widely used in cell biology to visualize the intracellular localization of proteins, to assess protein expression levels, to study protein-protein interactions, to track cellular processes (e.g., mitosis, apoptosis), and in diagnostics for identifying specific cellular markers (e.g., in cancer diagnosis or detecting infectious agents).

Flashcard #89
Term: What are the key advantages of using Immunofluorescence (IF)?
Definition: The key advantages include:

• High Specificity: Relies on specific antibody-antigen binding for precise localization.

• High Resolution: Can resolve subcellular structures and protein localization (depending on the microscope).

• Multiplexing: Multiple proteins can be simultaneously visualized using different fluorescent labels.

• Contextual Information: Provides spatial information about protein distribution in situ within cells or tissues.

Flashcard #90
Term: What are the major limitations or challenges of Immunofluorescence (IF)?
Definition: The major limitations include:

• Antibody Quality: Requires highly specific and validated antibodies; non-specific antibodies can lead to false positives.

• Autofluorescence: Tissue or cellular autofluorescence can interfere with signal detection.

• Fixation Artifacts: Sample fixation can sometimes alter antigenicity or cellular morphology.

• Quantification Challenges: Accurate absolute quantification of protein levels can be difficult due to variability in staining and imaging conditions.

Flashcard #91
Term: Why is Immunofluorescence (IF) considered an essential technique?
Definition: Immunofluorescence is considered an essential technique because it offers a direct and highly visual method to localize and visualize specific proteins within the complex environment of cells and tissues. This capability is fundamental for understanding protein function, cellular organization, and disease mechanisms, providing critical spatial information that other techniques might miss.

Flashcard #92
Term: What specific type of information does Immunofluorescence (IF) reveal about proteins?
Definition: Immunofluorescence reveals the subcellular localization, distribution, and relative abundance of specific proteins or antigens within cells or tissues. It can show if a protein is in the nucleus, cytoplasm, membrane, or specific organelles, and how its location or expression changes under different conditions, providing insights into its functional role.

Flashcard #93
Term: What is ELISA (Enzyme-Linked Immunosorbent Assay)?
Definition: ELISA (Enzyme-Linked Immunosorbent Assay) is a plate-based immunoassay technique used for detecting and quantifying soluble proteins, peptides, antibodies, hormones, and other analytes in liquid samples. It utilizes a solid-phase enzyme immunoassay, where an antigen or antibody is immobilized on a microplate surface and detected via an enzyme-linked antibody that produces a detectable signal (typically colorimetric) proportional to the amount of analyte present.

Flashcard #94
Term: What are the common steps involved in ELISA (Enzyme-Linked Immunosorbent Assay)?
Definition: The common steps involved in a typical 'sandwich' ELISA include:

1. Plate Coating: A capture antibody specific to the target antigen is coated onto the wells of a microplate.

2. Blocking: Unbound sites on the plate are blocked to prevent non-specific binding.

3. Sample Addition: The sample containing the target antigen is added and incubated, allowing the antigen to bind to the capture antibody.

4. Detection Antibody Addition: A detection antibody, also specific to the antigen (but binding to a different epitope), is added. This antibody may be enzyme-conjugated or detected by an enzyme-conjugated secondary antibody.

5. Substrate Addition: A substrate for the enzyme is added, which is converted into a colored, fluorescent, or luminescent product.

6. Signal Detection: The intensity of the signal (e.g., absorbance for colorimetric assays) is measured by a plate reader, which is directly proportional to the amount of antigen in the sample.

Flashcard #95
Term: In what context is ELISA (Enzyme-Linked Immunosorbent Assay) commonly used?
Definition: ELISA is widely used in clinical diagnostics for detecting antibodies against infectious agents (e.g., HIV, Lyme disease), for measuring hormone levels (e.g., thyroid hormones), for detecting tumor markers in cancer screening, and in research for quantifying cytokines or other proteins in biological samples.

Flashcard #96
Term: What are the key advantages of using ELISA (Enzyme-Linked Immunosorbent Assay)?
Definition: The key advantages include:

• High Sensitivity: Can detect analytes at very low concentrations (picomolar range).

• High Specificity: Relies on highly specific antibody-antigen binding.

• Quantification: Provides quantitative measurements of target analyte concentration.

• Throughput: Plate-based format allows for high-throughput screening of many samples simultaneously.

• Relatively Cost-Effective: Compared to some other quantification methods for proteins.

Flashcard #97
Term: What are the major limitations or challenges of ELISA (Enzyme-Linked Immunosorbent Assay)?
Definition: The major limitations include:

• Antibody Availability/Quality: Requires specific and high-quality antibodies; poor antibodies can lead to false results.

• Cross-Reactivity: Potential for antibodies to bind to non-target analytes, leading to false positives.

• Sample Matrix Effects: Components in complex biological samples can interfere with the assay.

• Dynamic Range: Limited dynamic range compared to some other methods, requiring sample dilution for high concentrations.

• Does not Provide Molecular Weight Information: Unlike western blot or mass spectrometry.

Flashcard #98
Term: Why is ELISA (Enzyme-Linked Immunosorbent Assay) considered an essential technique?
Definition: ELISA is considered an essential technique because it offers a highly sensitive, specific, and quantitative method for detecting and measuring a wide array of biomolecules in complex biological samples. Its plate-based format enables high-throughput analysis, making it indispensable for clinical diagnostics, therapeutic drug monitoring, and research requiring precise quantification of proteins and other analytes.

Flashcard #99
Term: What specific type of information does ELISA (Enzyme-Linked Immunosorbent Assay) reveal about analytes?
Definition: ELISA reveals the presence and concentration (quantitative) of specific antigens, antibodies, or other soluble analytes in a liquid sample. It tells you how much of a particular molecule is present, allowing for diagnosis (e.g., infection status), monitoring (e.g., therapeutic drug levels), or research quantification.

Flashcard #100
Term: What is Mass Spectrometry (MS)?
Definition: Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio (m/z) of ions. It is widely used in proteomics to identify and quantify proteins and peptides, elucidate their primary structures, and characterize post-translational modifications. By precisely measuring molecular weights, MS provides powerful insights into the composition and characteristics of molecules in a sample.

Flashcard #101
Term: What are the common steps involved in Mass Spectrometry (MS) for protein analysis?
Definition: The general steps involved in mass spectrometry for protein analysis include:

1. Sample Ionization: Molecules in the sample are converted into gas-phase ions (e.g., by electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI)).

2. Mass Analysis: The ions are separated in a vacuum based on their mass-to-charge ratio (m/z) by a mass analyzer (e.g., quadrupole, time-of-flight (TOF), Orbitrap).

3. Detection: The separated ions are detected, and their abundance is recorded.

4. Data Analysis: The resulting mass spectrum, which plots ion abundance against m/z, is analyzed to identify molecules by matching experimental m/z values to theoretical values in databases (for protein identification after enzymatic digestion into peptides) or to determine their molecular weight or modifications.

Flashcard #102
Term: In what context is Mass Spectrometry (MS) commonly used?
Definition: MS is broadly used in proteomics for protein identification and quantification (e.g., in differential expression studies), for mapping post-translational modifications (PTMs) like phosphorylation or glycosylation, in clinical diagnostics for biomarker discovery and detection, in drug discovery for target identification and drug metabolism studies, and in metabolomics for small molecule analysis.

Flashcard #103
Term: What are the key advantages of using Mass Spectrometry (MS)?
Definition: The key advantages include:

• High Specificity and Sensitivity: Capable of identifying and quantifying molecules with high precision and detecting analytes at very low concentrations.

• Broad Application Range: Applicable to a wide variety of molecules, from small metabolites to large proteins.

• Post-Translational Modification Analysis: Excellent for characterizing PTMs, which are crucial for protein function.

• Multiplexing and Throughput: Advanced MS platforms can analyze many samples or components simultaneously.

Flashcard #104
Term: What are the major limitations or challenges of Mass Spectrometry (MS)?
Definition: The major limitations include:

• Sample Preparation Complexity: Often requires extensive sample preparation (e.g., protein digestion for proteomics) that can introduce biases or losses.

• Ion Suppression/Matrix Effects: Components in complex biological samples can interfere with ionization, affecting quantification.

• High Cost and Expertise: MS instruments are expensive, and their operation and data analysis require specialized expertise.

• Dynamic Range Challenges: While sensitive, detecting extremely low abundance proteins alongside highly abundant ones can be challenging.

Flashcard #105
Term: Why is Mass Spectrometry (MS) considered an essential technique?
Definition: Mass spectrometry is considered an essential technique because it provides unparalleled capabilities for identifying, quantifying, and characterizing molecules based on their exact mass. In proteomics, it enables the comprehensive analysis of protein expression, modifications, and interactions on a global scale, fundamental for understanding cellular processes, disease mechanisms, and drug action, thereby driving advancements in biology and medicine.

Flashcard #106
Term: What specific type of information does Mass Spectrometry (MS) reveal about proteins?
Definition: Mass spectrometry reveals precise molecular weights of analytes and, for proteins treated with proteases, the m/z of their constituent peptides. This information is used to:

• Identify Proteins: By matching peptide masses to protein databases.

• Quantify Proteins: By comparing signal intensities of peptides across samples.

• Characterize Post-Translational Modifications: By detecting changes in molecular weight due to additions or cleavages.

• Determine Amino Acid Sequences: Through tandem MS (MS/MS) fragmentation pattern analysis.

Flashcard #107
Term: What is FACS (Fluorescence-Activated Cell Sorting)?
Definition: FACS (Fluorescence-Activated Cell Sorting) is a sophisticated flow cytometry technique that allows for the rapid, objective, and quantitative analysis and physical separation of individual cells from a heterogeneous mixture based on their unique light-scattering properties and fluorescence characteristics. Cells are labeled with fluorescent markers (antibodies or dyes) and then passed one by one through a laser beam; detected signals are used to sort them into distinct populations.

Flashcard #108
Term: What are the common steps involved in FACS (Fluorescence-Activated Cell Sorting)?
Definition: The common steps involved in FACS include:

1. Cell Suspension Preparation: Cells are dissociated from tissue or culture and prepared into a single-cell suspension. They are often stained with fluorescent antibodies or dyes.

2. Hydrodynamic Focusing: The single-cell suspension is guided by sheath fluid to flow as a single stream, ensuring cells pass one by one through the laser.

3. Laser Excitation and Detection: As each cell passes a laser beam, it scatters light (forward and side scatter, indicating size and granularity) and emits fluorescence (from labeled markers). Detectors convert these signals into electronic data.

4. Droplet Formation and Charging: The stream is vibrated to create uniform droplets, with individual cells encapsulated in droplets. Based on the real-time analysis of scatter and fluorescence, some droplets receive an electric charge (positive or negative).

5. Deflection and Sorting: Charged droplets pass through an electric field, which deflects them into different collection tubes, allowing for the isolation of specific cell populations.

Flashcard #109
Term: In what context is FACS (Fluorescence-Activated Cell Sorting) commonly used?
Definition: FACS is indispensable in immunology for isolating specific immune cell subsets (e.g., T cells, B cells) for downstream analyses, in cancer research for identifying and purifying cancer stem cells or tumor-infiltrating lymphocytes, in stem cell biology for enriching specific progenitor cells, and for separating cells based on reporter gene expression (e.g., GFP).

Flashcard #110
Term: What are the key advantages of using FACS (Fluorescence-Activated Cell Sorting)?
Definition: The key advantages include:

• High Speed: Can analyze and sort thousands of cells per second.

• High Purity: Allows for the isolation of highly pure cell populations based on multiple parameters.

• Quantitative Analysis: Provides quantitative data on cell size, granularity, and fluorescence intensity of individual cells.

• Multiparametric Analysis: Can simultaneously assess multiple cellular characteristics (e.g., co-expression of several surface markers).

• Viable Cells: Cells remain viable after sorting, allowing for downstream functional studies.

Flashcard #111
Term: What are the major limitations or challenges of FACS (Fluorescence-Activated Cell Sorting)?
Definition: The major limitations include:

• High Cost and Complexity: Requires expensive instruments and trained personnel for operation and maintenance.

• Sample Preparation: Requires single-cell suspension; clumping or debris can clog the system.

• Cell Stress: The sorting process can expose cells to stress (e.g., shear stress, pressure), potentially affecting their viability or function.

• Off-Target Cells: Small populations can be missed if the analysis gates are not set properly; requires careful compensation for spectral overlap.

Flashcard #112
Term: Why is FACS (Fluorescence-Activated Cell Sorting) considered an essential technique?
Definition: FACS is considered an essential technique because it uniquely combines rapid, high-throughput analysis with the ability to physically separate viable cell populations based on complex fluorescent and light-scattering properties. This capability is critical for dissecting cellular heterogeneity, enriching rare cell types, and exploring the characteristics of specific cell populations, driving advancements in immunology, stem cell research, and cancer biology.

Flashcard #113
Term: What specific type of information does FACS (Fluorescence-Activated Cell Sorting) reveal about cells?
Definition: FACS reveals the heterogeneity of a cell population, indicating the proportion and characteristics (size, granularity, and expression of specific surface or intracellular markers) of different cell subsets. Crucially, it isolates specific viable cell populations for further study, providing information on the functional relevance of these different cell types.

Flashcard #114
Term: What is Immunohistochemistry (IHC)?
Definition: Immunohistochemistry (IHC) is a powerful laboratory technique that uses specific antibodies to selectively visualize and localize antigens (proteins) in thin sections of biological tissue. By conjugating antibodies with an enzyme (e.g., horseradish peroxidase) or a fluorescent dye, IHC allows for the precise detection and spatial distribution of target proteins within the morphological context of the tissue architecture, aiding in disease diagnosis and research.

Flashcard #115
Term: What are the common steps involved in Immunohistochemistry (IHC)?
Definition: The common steps involved in IHC include:

1. Tissue Preparation: Tissue samples (e.g., biopsies) are fixed (e.g., with formalin), embedded in paraffin, and cut into thin sections. Alternatively, frozen sections can be used.

2. Antigen Retrieval: For formalin-fixed paraffin-embedded (FFPE) tissues, heat or enzymatic treatment is often performed to unmask antigens obscured by fixation.

3. Blocking: Non-specific binding sites in the tissue are blocked (e.g., with serum).

4. Primary Antibody Incubation: The tissue section is incubated with a primary antibody that specifically binds to the target antigen.

5. Washing: Unbound primary antibody is rinsed away.

6. Secondary Antibody Incubation: For indirect IHC, an enzyme- or fluorophore-conjugated secondary antibody (which binds to the primary antibody) is added. For direct IHC, the primary antibody itself is conjugated, and this step is skipped.

7. Detection: For enzyme-based IHC, a chromogenic substrate is added, producing a colored precipitate visible under a light microscope. For fluorescence-based IHC, a fluorescent signal is directly visualized.

8. Counterstaining and Mounting: Cells are counterstained (e.g., with hematoxylin) to provide morphological context, and slides are mounted for microscopy.

Flashcard #116
Term: In what context is Immunohistochemistry (IHC) commonly used?
Definition: IHC is a cornerstone in diagnostic pathology for classifying tumors (e.g., identifying breast cancer subtypes based on ER/PR/HER2 expression), determining prognosis, and guiding therapy decisions. In research, it's used to study protein expression patterns in disease progression, embryonic development, and to localize specific cell types within complex tissues.

Flashcard #117
Term: What are the key advantages of using Immunohistochemistry (IHC)?
Definition: The key advantages include:

• Preservation of Tissue Morphology: Allows for the visualization of protein expression in situ within the intact tissue architecture.

• High Specificity: Relies on specific antibody-antigen binding.

• Diagnostic Power: Provides crucial diagnostic and prognostic information in clinical pathology.

• Multiplexing (with limitations): Can visualize multiple markers using different colored chromogens or fluorophores.

Flashcard #118
Term: What are the major limitations or challenges of Immunohistochemistry (IHC)?
Definition: The major limitations include:

• Antibody Quality: Requires highly validated and specific antibodies; non-specific antibodies lead to false positives.

• Fixation Artifacts: Tissue fixation and processing can affect antigenicity and staining quality.

• Autofluorescence (for IF-IHC): Background autofluorescence in tissues can obscure signals.

• Quantification Challenges: Semiquantitative interpretation can be subjective; precise quantification is generally difficult compared to plate-based assays.

• Single-Section Analysis: Provides information only from the specific section analyzed, not the entire tissue.

Flashcard #119
Term: Why is Immunohistochemistry (IHC) considered an essential technique?
Definition: IHC is considered an essential technique because it bridges molecular biology with histology, allowing for the visualization of specific protein expression directly within the anatomical context of tissues. This capability is fundamental for understanding disease pathogenesis, classifying tumors, and guiding clinical decisions, providing invaluable insights into protein localization and abundance in situ.

Flashcard #120
Term: What specific type of information does Immunohistochemistry (IHC) reveal about proteins in tissues?
Definition: IHC reveals the presence, cellular and subcellular localization, and relative abundance of specific proteins within tissue sections. It shows which cell types express a particular protein, where it's located within those cells, and can indicate changes in expression levels between healthy and diseased states, providing critical diagnostic and research information within the tissue's morphological context.

Flashcard #121
Term: What are Multiplex Immunoassays?
Definition: Multiplex immunoassays are advanced immunoassay technologies that enable the simultaneous detection and quantification of multiple distinct analytes (e.g., proteins, cytokines, antibodies) from a single biological sample. Unlike traditional single-plex ELISA, these assays utilize bead-based or planar array technologies with different labels or spatial separation to differentiate and measure signals from numerous targets concurrently, significantly increasing throughput and conserving valuable sample volume.

Flashcard #122
Term: What are the common steps involved in Multiplex Immunoassays?
Definition: The common steps involved in bead-based multiplex immunoassays (e.g., Luminex) include:

1. Capture Bead Incubation: Magnetic beads, each uniquely identifiable by a spectral address (color/fluorescence tag) and coated with a specific capture antibody for one analyte, are mixed and incubated with the sample.

2. Detection Antibody Incubation: A cocktail of biotinylated detection antibodies, each specific to one of the captured analytes, is added.

3. Streptavidin-Phycoerythrin (SAPE) Incubation: A fluorochrome-labeled streptavidin (which binds to biotin) is added to generate a fluorescent signal proportional to the analyte concentration.

4. Reading on Flow Cytometer: Beads are passed through a flow cytometer. Lasers identify each bead (by its spectral address) and quantify the reporter fluorescence (from SAPE) on its surface, allowing simultaneous measurement of multiple analytes.

5. Data Analysis: Software generates concentration data for each analyte based on standard curves.

Flashcard #123
Term: In what context are Multiplex Immunoassays commonly used?
Definition: Multiplex immunoassays are widely used in biomarker discovery and validation (e.g., for inflammatory markers, cancer biomarkers), in systems biology to profile cytokine or chemokine expression in disease, in vaccine development to assess immune responses to multiple antigens, and in drug development for pharmacokinetic and pharmacodynamic studies.

Flashcard #124
Term: What are the key advantages of using Multiplex Immunoassays?
Definition: The key advantages include:

• High Throughput: Simultaneously quantify many analytes from a single sample, saving time and resources.

• Sample Conservation: Requires less sample volume compared to running multiple single-plex assays.

• Cost-Effective: Can be more cost-effective than numerous individual ELISA assays for large panels of analytes.

• Comprehensive Data: Provides a broader snapshot of biological pathways or immune responses.

Flashcard #125
Term: What are the major limitations or challenges of Multiplex Immunoassays?
Definition: The major limitations include:

• Cross-Reactivity/Interference: Potential for interactions between antibodies or analytes, leading to false positives or inaccurate quantification.

• Complexity of Development: Developing and optimizing multiplex assays is more complex than single-plex assays.

• Dynamic Range Differences: Different analytes within the same assay might have varying dynamic ranges of detection.

• Instrument Cost: Specialized plate readers or flow cytometers are required for analysis.

Flashcard #126
Term: Why are Multiplex Immunoassays considered essential techniques?
Definition: Multiplex immunoassays are considered essential techniques because they dramatically increase the efficiency of biomarker discovery and validation by enabling the simultaneous quantification of numerous targets from a single, often limited, biological sample. This capability is critical for understanding complex biological systems, identifying disease signatures, and accelerating research in areas like immunology, oncology, and personalized medicine.

Flashcard #127
Term: What specific type of information do Multiplex Immunoassays reveal about analytes?
Definition: Multiplex immunoassays reveal the qualitative presence and quantitative concentration of multiple specific analytes (e.g., proteins, cytokines, antibodies) simultaneously within a single sample. This provides a comprehensive profile of various markers involved in a biological process or disease state, allowing for the identification of complex biomarker signatures or panel-based diagnostics.

Flashcard #128
Term: What is Fluorescence Microscopy?
Definition: Fluorescence microscopy is an optical microscopy technique that uses fluorescence and phosphorescence instead of, or in addition to, reflection and absorption to study properties of organic or inorganic substances. It utilizes specific fluorophores (fluorescent dyes or proteins) that absorb light at one wavelength and emit it at a longer wavelength, allowing for the visualization of labeled molecules or structures within cells and tissues with high specificity and sensitivity.

Flashcard #129
Term: What are the common steps involved in Fluorescence Microscopy?
Definition: The common steps involved in fluorescence microscopy include:

1. Sample Preparation and Staining: The biological sample (cells or tissue sections) is prepared (e.g., fixed, permeabilized) and stained with fluorescent labels (e.g., fluorescent dyes, fluorescently tagged antibodies, or genetically encoded fluorescent proteins like GFP) that bind specifically to the molecule or structure of interest.

2. Light Source and Excitation: The prepared sample is placed on a microscope stage. A high-intensity light source (e.g., mercury lamp, LED, laser) emits light at a specific excitation wavelength, which is directed onto the sample through an excitation filter.

3. Fluorophore Emission: The fluorophores in the sample absorb the excitation light and then emit light at a longer, characteristic emission wavelength.

4. Emission Filter and Detection: The emitted fluorescent light passes through an emission filter, which blocks scattered excitation light, allowing only the fluorescent signal to reach the detector (e.g., camera, eyepiece).

5. Image Capture and Analysis: The detector captures the fluorescent image, which can then be processed and analyzed to visualize and quantify the labeled structures.

Flashcard #130
Term: In what context is Fluorescence Microscopy commonly used?
Definition: Fluorescence microscopy is extensively used in cell biology to visualize subcellular organelles, protein localization and dynamics, cell morphology, and cellular processes (e.g., endocytosis, cell division). It's also used in pathology for diagnostic imaging and in neurobiology to map neuronal connections or track cellular events in living organisms.

Flashcard #131
Term: What are the key advantages of using Fluorescence Microscopy?
Definition: The key advantages include:

• High Specificity: Allows for selective visualization of target molecules against a dark background, due to specific labeling.

• High Sensitivity: Can detect low concentrations of fluorescently labeled molecules.

• Multiplexing: Multiple fluorophores with distinct emission spectra can be used simultaneously to visualize several targets.

• Live-Cell Imaging: Enables dynamic studies of cellular processes in real-time (with appropriate setup).

• Contextual Information: Provides spatial information about molecular and cellular organization.

Flashcard #132
Term: What are the major limitations or challenges of Fluorescence Microscopy?
Definition: The major limitations include:

• Photobleaching: Fluorophores can irreversibly lose their fluorescence due to prolonged exposure to excitation light, limiting observation time.

• Autofluorescence: Background autofluorescence from biological samples can interfere with specific signals.

• Phototoxicity: High-intensity light can be toxic to living cells, especially during long-term imaging.

• Limited Penetration Depth: Light scattering typically limits imaging to thin samples or superficial layers for thick samples.

• Maintenance and Cost: Requires specialized, often expensive, equipment and skilled operation.

Flashcard #133
Term: undefined
Definition: Fluorescence microscopy is considered an essential technique because it offers a highly specific and sensitive way to visualize and study molecules and structures within the complex, living environment of cells and tissues. Its ability to enable dynamic live-cell imaging and multiplex labeling provides fundamental insights into cellular processes, protein localization, and molecular interactions, which are