Bio 251 Spring 2025 Final Exam Study Guide
Exam Overview
Format: 1 hour 50 minutes, with both parts given simultaneously.
Versions: Multiple versions of the exam will be available.
Point Distribution:
Multiple Choice: 100 points (20 new material, 30 cumulative).
Short Answer: 50 points (2 new material, 2 cumulative).
Content: Includes multiple-choice questions, short answer questions, and drawing/labeling exercises.
Study Tips
Use lecture PowerPoints and textbook figures to identify important topics.
Label and explain key figures; consolidate related figures into overall diagrams.
Draw simple representations of figures and label all parts using vocabulary lists.
Write down information to aid in answering questions; avoid doing all work mentally.
Collaborate with peers to gain different perspectives.
Attend student hours for clarification and testing understanding.
Core Concepts
From Autoradiogram to Protein
Sequencing: Read autoradiograms from bottom to top (A, T, G, C lanes).
Template Strand: The obtained sequence is complementary to the template strand.
Transcription: Produces the coding strand, identical to the template strand except T is replaced by U.
Translation: mRNA is divided into codons, starting from the start codon, AUG.
Mutation Impact on Protein
Loss of Function: Protein is non-functional or has reduced function; often recessive (e.g., cystic fibrosis).
Gain of Function: New or enhanced protein activity, or activity at inappropriate times/tissues; typically dominant.
Neutral Mutations: Change amino acid sequence without significantly altering protein function.
Missense Mutations: Change one amino acid in the protein, affecting function depending on the amino acid's role.
Nonsense Mutations: Introduce a premature stop codon, typically making the protein non-functional.
Frameshift Mutations: Insertions or deletions not in multiples of 3, shifting the reading frame.
Silent Mutations: Do not change the amino acid sequence due to codon redundancy but can affect gene expression, protein folding, or splicing.
Mendelian Genetics
Monohybrid Cross: One trait with two alleles (e.g., RR x rr).
Individuals have two alleles that separate during gamete formation.
Dihybrid Cross: Two traits, two genes with two alleles (e.g., RRYY x rryy).
Independent assortment: alleles at different loci segregate independently during gamete formation.
Complete Dominance: Heterozygote phenotype is identical to one homozygote (e.g., RR and Rr show the same phenotype).
Incomplete Dominance: Heterozygote phenotype is intermediate between two homozygotes (e.g., RR (red) x rr (white) creates Rr (pink)).
Codominance: Heterozygote expresses traits from both homozygotes (e.g., a white cow with brown spots).
Multiple Alleles
More than two alleles exist in a single gene locus within a population.
Each individual inherits two alleles, but more than two are possible in the population.
Example: blood group in humans.
Number of genotypes possible: (n(n+1))/2, where n = number of alleles.
Epistasis
One gene's effect is masked or modified by another gene at a different locus.
Masking gene: epistatic.
Masked gene: hypostatic.
Experiment Design for Gene Function
Determine the function of gene X (suspected to be involved in cell division).
Use CRISPR-Cas9 gene editing to knock out gene X in cultured mammalian cells.
Transfer cells with CRISPR-Cas9, select knockout clones via sequencing.
Compare growth rate, cell cycle progression, and apoptosis markers to control-transfected cells.
Analyze and interpret phenotype differences.
Gene Expression Regulation in Prokaryotes
Negative vs. Positive Control:
Negative: Repressor binds to the operator to block transcription.
Positive: Activator enhances RNA polymerase binding.
Operon Control:
Inducible operons: Genes are off by default, turned on by an inducer (e.g., lac operon).
Repressible operons: Genes are on by default, turned off by a corepressor (e.g., trp operon).
Attenuation: Early termination of transcription based on secondary mRNA structures.
Gene Expression Regulation in Eukaryotes
Chromatin Modification:
Histone acetylation: Opens chromatin, activates transcription.
DNA methylation: Silences genes (epigenetic regulation).
Transcription Factors: Bind to enhancers, silencers, and promoters to regulate initiation.
RNA Processing: Alternative splicing creates multiple proteins from one gene; RNA editing and mRNA capping/tailing affect transcript stability and translation.
RNA Interference: siRNA and miRNA can bind mRNA and block translation or cause degradation.
Translational and Post-Translational Control: Regulation of translation initiation, protein modification (phosphorylation, ubiquitination) affects activity and degradation.
Cell Division and Genetic Material Inheritance
DNA Replication: Occurs during the S phase of the cell cycle before cell division.
Mitosis: Produces two genetically identical daughter cells; chromosomes duplicate and segregate equally; used for growth, tissue repair, and asexual reproduction.
Mitosis Steps
Prophase: Chromosomes condense and become visible, mitotic spindles begin to form from centrosomes, and the nucleus disappears.
Prometaphase: The nuclear envelope breaks down, and spindle microtubules attach to kinetochores on centromeres.
Metaphase: Chromosomes align along the metaphase plate; each chromosome is attached to spindle fibers from opposite poles.
Anaphase: Sister chromatids separate and move toward opposite poles; each chromatid is now an individual daughter chromosome.
Telophase: Chromosomes decondense, the nuclear envelope reforms around each set of chromosomes, and the spindle disassembles.
Cytokinesis: The cytoplasm divides, forming two separate daughter cells.
Meiosis: Produces 4 non-identical haploid cells (gametes); includes two divisions: meiosis I (homologous chromosomes separate) and meiosis II (sister chromatids separate).
Meiosis Steps
Meiosis I:
Prophase I: Most complex phase; synapsis and crossing over between homologous chromosomes; chromosomes begin to condense, homologous chromosomes pair, and crossing over occurs; chromosomes are fully condensed and ready for alignment.
Metaphase I: Homologous chromosome pairs line up at the metaphase plate, and each pair attaches to spindle fibers from opposite poles.
Anaphase I: Homologous chromosomes separate (sister chromatids stay together).
Telophase I and Cytokinesis: Nuclear membranes may reform, and the cell divides into two haploid cells.
Meiosis II:
Prophase II: A new spindle apparatus forms, and chromosomes condense again (still with 2 sister chromatids).
Metaphase II: Chromosomes align at the metaphase plate.
Anaphase II: Sister chromatids separate and move to opposite poles.
Telophase II and Cytokinesis: The nuclear envelope reforms, and the cytoplasm divides, resulting in 4 genetically distinct haploid cells.
Introduces genetic variation through crossing over (prophase I) and independent assortment (metaphase I).
Errors in Processes Resulting in Disease
Errors in DNA Replication → Mutations: Point mutations, insertions, deletions, or copy number variations can lead to genetic disorders or cancer.
Chromosomal Segregation Errors (Meiosis): Nondisjunction leads to an abnormal number of chromosomes, e.g., Down syndrome (extra copy of chromosome 21) and Turner syndrome (XO) or Klinefelter syndrome (XXY).
Errors in Transcription or RNA Processing: Mutations that affect splicing, promoter regions, or regulatory elements can lead to incorrect or absent protein production.
Protein Misfolding or Malfunction: Missense mutations or post-translational errors can produce misfolded proteins (e.g., sickle cell disease).
Failure of Cell Cycle Control → Cancer: Mutations in genes that regulate cell division (e.g., p53) lead to uncontrolled proliferation, which accumulates through replication errors or environmental exposures (UV, carcinogens).
Molecular Analysis and Biotechnology
Restriction Enzymes
Bacterial enzymes that cut DNA at specific sequences; serve as a defense system in bacteria by destroying invading viral DNA.
Recognition Site Binding: Each restriction enzyme recognizes a specific DNA sequence, typically 4-8 bp long and the same forward and backward.
DNA Cleavage: The enzyme cuts both DNA strands at or near the recognition site, creating either sticky ends (overhanging single-stranded ends) or blunt ends (straight cuts across both strands).
Resulting Fragments: The cut DNA can be joined with other DNA fragments having compatible ends using DNA ligase.
Nucleases
Restriction Endonucleases (Restriction Enzymes): Cut DNA within a specific sequence, internal cleavage at recognition sites, to generate defined DNA fragments for cloning or analysis.
Exonucleases: Remove nucleotides one at a time from the end of a DNA strand, progressive digestion from the 5’ or 3’ end, useful in deleting specific DNA regions, generating single-stranded templates, or preparing blunt ends.
CRISPR-Cas System
A genome editing tool derived from bacterial immune defense mechanisms allows for precise target changes to DNA in living cells.
Mechanism:
Design a gRNA that matches the DNA sequence.
Cas9 binds the gRNA and scans DNA for the target sequence next to a PAM site.
Cas9 cuts both DNA strands at the target site.
The cell repairs the break using non-homologous end joining or homology-directed repair.
Modifications:
Cas9 nickase: Cuts only one DNA strand → reduces off-target effects.
dCas9 (dead): Mutated Cas9 that binds DNA without cutting → used for gene regulation.
CRISPR: dCas9 fused with an activator (a) or repressor (i) to control gene expression.
Base editors: Fuse Cas9 with enzymes to convert A→T or C→G without cutting DNA.
Prime editing: Search and replace using Cas9 and reverse transcriptase.
Cas12 and Cas13 systems: Other enzymes that target DNA (Cas12) or RNA (Cas13) for broader uses.
Advantages:
Target specificity: CRISPR uses a guide RNA that can be precisely programmed to target virtually any DNA sequence.
Efficiency: High success rate in introducing target edits compared to older methods.
Simplicity: Easier to design and use – requires only Cas protein and a gRNA.
Versatility: Works in a wide range of organisms and cell types (bacteria to mammals).
Multiplexing ability: Can edit multiple genes at once by introducing multiple gRNAs.
Broad applications: Used for gene knockouts, insertions, base editing, epigenetic regulation, diagnosis, and more.
Cost-effective: Cheaper than older genome-editing technologies.
Limitations:
Off-target effects: Cas enzymes may cut DNA at sites that are similar, but not identical, to the target, causing unwanted mutations.
PAM sequence dependency: Editing is restricted to DNA sequences near a PAM site.
Delivery challenges: Delivering CRISPR components into some cell types can be difficult.
Immunogenicity: Cas proteins can trigger immune responses in humans.
Repair pathway limitations: Relies on cellular DNA repair mechanisms; non-homologous end joining is error-prone, while homology-directed repair is less efficient.
Mosaicism: In multicellular organisms (like embryos), not all cells may be edited, leading to inconsistent outcomes.
Ethical concerns: Can create heritable genetic changes with long-term consequences.
DNA Fragment Separation and Visualization
Gel Electrophoresis: DNA fragments are loaded into wells of a gel matrix (agarose), then an electric current is applied; DNA moves toward the positive electrode, smaller fragments migrate faster and farther than larger ones; DNA fragments separate by size.
Capillary Electrophoresis: Used in automated DNA sequencing; DNA fragments migrate through a thin capillary filled with a gel-like polymer; a fluorescent dye attached to DNA enables laser detection at high resolution.
Southern Blotting: After gel electrophoresis, DNA is transferred to a membrane and hybridized with a labeled probe; allows detection of specific DNA sequences.
PCR (Polymerase Chain Reaction), Requirements and Steps
Requirements:
Template DNA
Primers
DNA polymerase
dNTPs (A, T, C, G)
Buffer solution
Thermal cycler
Steps:
Denaturation (94-96 °C): Heat the reaction to separate double-stranded DNA into single strands.
Annealing (50-65 °C): Cool the reaction to allow primers to bind to complementary sequences on the single-stranded DNA.
Extension (72 °C): DNA polymerase adds nucleotides to the 3’ ends of the primers to synthesize new DNA strands.
After each cycle, the number of DNA copies doubles; after ~30 cycles, over 1 billion copies of the target sequence can be generated.
Gene Cloning Process Requirements and Steps
Requirements: DNA fragment, cloning vector, restriction enzymes, DNA ligase, host cells.
Steps:
Isolate and cut the DNA: The gene of interest and plasmid vector are cut with the same restriction enzymes, creating compatible sticky ends.
Ligation: The gene and vector are mixed and joined using DNA ligase, forming recombinant DNA.
Transformation: The recombinant DNA is introduced into competent bacterial cells via heat shock or electroporation.
Selection: Bacteria are grown on selective media to identify those that took up the plasmid.
Replication and expression: Inside the host, the plasmid is replicated, producing many copies. The gene may also be transcribed and translated into protein.
DNA Sequencing Methods
Sanger Sequencing (Dideoxy or Chain Termination Method):
Uses DNA polymerase, a primer, template DNA, and a mix of normal nucleotides (dNTPs) and modified nucleotides (ddNTPs) that lack a 3’-OH group.
When a ddNTP is incorporated, chain elongation stops because no further nucleotides can be added.
Generates DNA fragments of various lengths.
Fragments are separated by size using gel or capillary electrophoresis.
Each ddNTP is labeled with a different fluorescent dye to identify the terminal base.
A fluorescence detector reads the sequence based on color and length.
Next-Generation Sequencing (NGS):
High throughput sequencing; sequences millions of DNA fragments in parallel.
DNA is fragmented and attached to adapters, then immobilized on a solid surface, amplified into clusters, and sequenced base-by-base using fluorescently labeled nucleotides.
A camera captures real-time base addition at each cluster location.
DNA Fingerprinting for Individual Identification
A technique used to identify an individual based on unique patterns in their DNA.
Analyzing short tandem repeats (STRs).
Short DNA sequences, 2-6 bp long, that are repeated in tandem at specific loci in the genome.
The number of repeats varies among individuals, making STR regions highly polymorphic.
The differences in repeat number form the basis of a person's genetic fingerprint.
Comparison of Forward and Reverse Genetics
Feature | Forward Genetics | Reverse Genetics |
|---|---|---|
Starting Point | Observed phenotype | Known gene/DNA sequence |
Goal | Identify the gene responsible for a trait | Determine the function of a specific gene |
Method | Induce mutations and screen for altered taits | Disrupt or modify the gene and observe the resulting phenotype |
Common Techniques | - Random mutagenesis - Linkage mapping - Gene identification | - Gene knockout |
Overexpression
Site directed mutagenesis |
| Time to Identify Gene | Usually longer | Faster, due to known gene |
| Usefulness | Ideal for discovering unknown genes | Ideal for analyzing known genes
Targeted Mutagenesis Techniques
Techniques used to introduce specific, intentional changes in the DNA sequence of a gene to study its function; alters known DNA sequences in precise locations.
Site-Directed Mutagenesis:
Introduces nucleotide changes at a defined site in a DNA molecule.
Change a single amino acid to study protein function.
Test effects of regulatory element changes.
CRISPR-Cas9-Mediated Mutagenesis:
A gene-editing system that uses Cas9 nuclease guided by an RNA molecule to cut DNA at a specific sequence.
Introduces knockouts, adds or corrects sequences via homology-directed repair.
Gene Knockout (Targeted Deletion):
Completely disables or deletes a gene to analyze its function by removing it from the genome.
To study gene essentiality and model human diseases in organisms.
Gene Knock-In (Insert):
A modified or foreign gene is inserted into a specific locus in the genome.
To study disease-causing mutations or add reporter genes to track gene expression.
Forward Genetics:
Start with an observable trait and work backward to identify the gene responsible.
Key Methods:
Mutagenesis: Introduce random mutations and screen for phenotypic changes.
Mapping and Cloning: Once a mutation is found, map its location and identify the gene.
Complementation Testing: Cross mutants to see if mutations are in the same gene or different ones.
Best for discovering unknown genes involved in biological processes.
Reverse Genetics:
Start with a known gene and disrupt or modify it to see what effect it has on the organism.
Techniques Include:
Gene Knockout: Entire gene is deleted or inactivated; observe what function is lost.
Gene Knockdown: Reduce gene expression using RNA interference or antisense; useful for essential genes where complete loss would be lethal.
Site-Directed Mutagenesis: Change specific DNA bases to alter amino acids or regulatory regions; analyze how mutations affect gene or protein function.
Transgenic Organism: Insert a gene into an organism to test overexpression or function in a new context; add tags like GFP to observe protein localization.
Gene Function Determination
Biochemical and molecular approaches
Protein Assays: Reveals enzymatic function, activity changes.
Reporter Genes: Reveals gene expression patterns.
Western Blot/ELISA: Reveal protein production and size.
Chromatin Immunoprecipitation: Reveals DNA binding sites for regulatory proteins.
Comparative and evolutionary approaches
Homology Analysis: Compare the gene to similar genes in other species.
If a gene is conserved, it likely has an essential function.
Gene Ontology and Databases: Use annotations from genome databases to predict function based on known genes.
Molecular Biotechnology Improvements to Human Life
Medical Contributions
Production of Therapeutic Proteins:
Insulin: Produced in E. coli using recombinant DNA, replacing animal-derived insulin.
Growth hormone, clotting factors, and vaccines.
Gene Therapy:
Faulty or missing genes are replaced or corrected in a patient's cells.
Used in the treatment of genetic disorders.
CRISPR-Based Therapies:
Precisely edit genes in human cells; trials are underway for sickle cell disease and some cancers.
Personalized Medicine:
Genetic profiling helps tailor drug treatments to individuals.
Disease Diagnosis:
PCR and molecular probes detect infections quickly and accurately; genetic testing identifies carriers of hereditary diseases.
Agricultural Contributions
Genetically Modified Crops:
Crops are engineered for insect resistance, herbicide tolerance, drought or salt tolerance, and nutritional enhancement.
Improved Animal Breeding:
Animals are engineered for higher milk production, leaner meat, and disease resistance.
Gene Editing in Plants and Animals:
CRISPR is used to develop disease-resistant pigs and non-browning mushrooms.
Biofactories:
Plants and animals are engineered to produce pharmaceuticals or industrial enzymes.
Key Terminology and Definitions
Recombinant DNA: DNA molecules formed by combining genetic material from two or more different sources, usually from different organisms, to create new genetic combinations that do not occur naturally.
Cutting DNA from two sources using restriction enzymes, joining the fragments using DNA ligase, inserting the recombinant molecule into a host organism for replication and expression.
Restriction Enzymes: Proteins that cut DNA at specific nucleotide sequences; can cut in two ways: blunt and sticky ends.
Restriction Sites: Each enzyme recognizes a specific sequence of bases (usually 4-8 bp) called a recognition site.
Usually a short, palindromic DNA sequence (4-8 bp long).
Palindromic: the sequences reads the same forward and backward on opposite strands. Example sequences: GAATTC, AAGCTT, GGATCC, CCCGGG, or CTGCAG.
Sticky Ends: Single-stranded overhangs of DNA that result when a restriction enzyme cuts DNA in a staggered fashion; can easily base-pair with complementary sequences; leave short, single-stranded overhangs that are complementary to each other; can stick to other DNA fragments with matching sequences.
Blunt Ends: DNA fragments with no overhangs—both strands of the DNA are cut straight across at the same nucleotide position; produced by restriction enzymes; produces flat, double-stranded ends with no single-stranded overhangs; less efficient but more versatile.
Engineered Nucleases: Artificially designed enzymes that can cut DNA at specific target sequences; combine a DNA-binding domain with a DNA-cleaving domain, allowing for precise genome edits; create double-stranded breaks in DNA, triggering cellular DNA repair mechanisms.
Zinc-Finger Nucleases (ZFNs):
Engineered DNA-cutting enzymes used for targeted genome editing.
Consist of two main parts: a DNA-binding domain made of zinc finger proteins that recognize specific DNA sequences and a DNA-cleaving domain, which cuts DNA once bound.
Transcription Activator-Like Effector Nucleases (TALENs):
Engineered proteins used for precise genome editing, similar to ZFNs.
Consist of two parts: a DNA-binding domain—derived from transcription activator-like effectors from plant pathogenic bacteria—and the FokI nuclease domain, which cuts DNA when dimerized.
Short, repetitive stretches of DNA.
CRISPR: Genetic editing technology, allowing for precise changes to the DNA of living organisms such as humans, plants, and animals.
Effector Complex: A group of proteins and molecules that work together to carry out the desired genetic modification after the CRISPR system has targeted a specific region of DNA.
Homologous Recombination: A natural process in cells where genetic material is exchanged between similar or identical DNA molecules; plays a critical role in DNA repair, especially during the process of repairing double-strand breaks; template DNA is provided, containing the desired genetic sequence that is homologous; this is then used as a guide to repair the break, swapping the broken DNA sequence with the template sequence, leading to insertion, deletion, or modification of DNA at the target site.
Non-Homologous End Joining: Directly joins the broken ends of DNA without the need for a template, making it a faster but less precise repair mechanism; repairs double-strand breaks in DNA.
Gel Electrophoresis: A laboratory technique used to separate and analyze DNA, RNA, or proteins based on their size, charge, and other properties; used for gene cloning, mutation analysis, DNA fingerprinting, and protein analysis.
Probe: A short, single-stranded sequence of nucleotides that is complementary to the sequence of interest in a sample; used to detect the presence of specific sequences of DNA, RNA, or proteins through hybridization or other detection methods; must be complementary to the sequence being detected.
Polymerase Chain Reaction (PCR): A series of temperature-driven steps that lead to the amplification of a specific DNA fragment.
Denaturation, annealing, elongation, repeat.
Components: DNA template, primers, Taq polymerase, nucleotides, buffer solution, magnesium ions.
Applications: DNA cloning, genetic testing, forensic analysis, pathogen detection, gene expression, environmental DNA detection, mutation detection.
Gene Cloning: The process of creating identical copies of a specific gene or DNA sequence; isolate, amplify, and study a particular gene or DNA fragment.
Cloning Vector: A small, self-replicating DNA molecule used to carry and replicate foreign DNA fragments within a host cell; serve as vehicles to introduce and maintain recombinant DNA inside the host organism for amplification or expression.
Linkers: DNA sequences that are used to facilitate the insertion of a DNA fragment into a cloning vector or between different pieces of DNA; commonly used in molecular cloning and recombinant DNA technology to add specific restriction enzyme sites to the ends of DNA fragments, which can then be inserted into vectors with complementary restriction sites.
Expression Vectors: Types of cloning vectors designed to drive the expression of a specific gene in a host cell; ensure that the inserted gene is not only cloned but also transcribed and translated to produce functional proteins.
Transformation: Foreign DNA is introduced into a cell, leading to the incorporation and expression of that DNA within the cell.
In Situ Hybridization: Used to detect specific nucleic acid sequences within a tissue or cell sample; involves the use of a labeled probe that is complementary to the target sequence of interest; the probe binds to its complementary sequence in the sample and produces a detectable signal that allows the researcher to locate and visualize the target nucleic acid in their natural context within tissues or cells.
Dideoxy Sequencing: A method used to determine the nucleotide sequence of DNA; relies on the selective incorporation of ddNTPs, which terminates DNA strand elongation, enabling the determination of the DNA sequence; DNA denaturation, primer binding, DNA synthesis, incorporation of ddNTPs, fragment lengths, separation by gel electrophoresis, detection.
Illumina Sequencing: Enables the rapid sequencing of large amounts of DNA at a much lower cost compared to dideoxy; massive parallelism (parallel sequencing of millions of DNA fragments at the same time), short read lengths (50-300bp), high accuracy, scalability.
DNA Fingerprinting: A technique used to identify individuals based on the unique characteristics of their DNA; takes advantage of the fact that each person has a distinct DNA sequence; widely used in forensic science, paternity testing, and genetic identity verification.
Forward Genetics: Begins with a phenotype of interest and then works to discover the underlying genetic basis through experimental techniques.
Identify the phenotype, Mutagenesis, Screen for mutants, Genetic mapping, gene identification, Validation.
Reverse Genetics: Starting with the gene itself and then analyzing the effects of manipulating it, works backward to understand its role in a particular biological process or trait.
Gene identification, gene knockout/knockdown, gene overexpression, observing phenotypic effects, functional validation.
Targeted Mutagenesis: The deliberate introduction of mutations into specific genes or regions of the genome in order to study their function; involves directing the mutagenesis process to a particular gene or DNA sequence of interest.
Site-Directed Mutagenesis: A technique used to introduce specific, targeted changes into a DNA sequence at a precise location; modify a particular gene by creating point mutations, insertions, or deletions at defined sites in the DNA; controlled, specific mutations.
Oligonucleotide-Directed Mutagenesis: A technique used to introduce specific mutations into a DNA sequence using synthetic oligonucleotides (short, single-stranded DNA molecules); allows modification of genes with high precision by introducing point mutations, insertions, or deletions at specific sites within a gene or genome.
Transgene: A gene that has been introduced into an organism's genome through genetic engineering techniques; typically derived from other species, but can come from the same species, depending on the desired modification; used to create genetically modified organisms (GMOs), which can be plants, animals, or microorganisms with altered genetic material.
Gene Therapy: A medical treatment that involves altering or replacing the genetic material within a person's cells to treat or prevent disease.
Key Concepts and Topics
Polymerase Chain Reaction (PCR): Allows for simplification of specific DNA sequences.
Human Genome Project (HGP): An effort to sequence the entire human genome, identifying 20k-25k genes in human DNA; provided the first complete map of human DNA.
CRISPR-Cas9: A gene-editing technology that allows scientists to make precise changes to the DNA of living organisms; uses a guide RNA to target specific DNA sequences, and the Cas9 protein cuts the DNA, enabling gene modification or gene replacement.
Next-Generation Sequencing (NGS): Allows for rapid and high-throughput sequencing of entire genomes or transcriptomes at a fraction of the cost and time.
CRISPR-Cas9 System Mechanics
Guide RNA (gRNA):
Complementary to a specific target DNA sequence.
Directs the Cas9 protein to the precise location on the DNA to be modified.
Consists of: Spacer sequence: Complementary to the target DNA. scaffold sequence: Binds to the Cas9 protein.
Cas9 Protein:
Binds to the gRNA.
Follows it to the target DNA sequence.
Makes a double-strand break in the DNA (DNA double-strand break - DSB).
Delivery into Cells:
gRNA and Cas9 protein are introduced into cells - use of electroporation, viral vector, and liposomes.
After DNA is cut, the cell tries to repair the break:
Non-homologous end joining.
Homology-directed repair
Homologous Recombination vs. Non-Homologous End Joining Comparison
Feature | Homologous Recombination | Non-Homologous End Joining |
|---|---|---|
Repair Mechanism | Uses homologous template for precise repair | Direct ligation of DNA ends (error-prone) |
Accuracy | Highly accurate | Error-prone (indels may occur) |
Speed | Slower (requires template and alignment) | Faster (direct end joining) |
Cell Cycle Phase | S and G2 phases (when sister chromatids are available) | G1 and throughout the cell cycle |
Genetic Stability | High (precise repair) | Low (mutations, frame-shifts may occur) |
Applications | Gene editing, gene replacement, precise repair | Gene knockout, DNA repair, emergency response |
Role in Genome Editing | Used for precise modifications (gene insertion) | Used for gene knockout (CRISPR-Cas9) |
Advantages of CRISPR-Cas9 System:
Precision and Specificity.
Efficiency.
Cost-Effectiveness.
Versatility.
Simplicity.
Potential for Therapeutic Applications and Large-Scale Use.
Limitations of CRISPR-Cas9
Off-Target Effects.
Incomplete or Inaccurate DNA Repair.
Delivery Challenges.
Ethical and Safety Concerns.
Limitations in Certain Cell Types.
Potential for Unintended Genetic Consequences.
CRISPR-Cas9 Modifications Overview:
High-Fidelity Cas9: Engineered to reduce off-target activity.
Dead Cas9 (dCas9): Mutant form lacking ability to cleave DNA; allows regulation of gene expression without permanent changes..
Base Editing: Uses dCas9 fused with a deaminase enzyme; enables precise point mutations.
Prime Editing: Direct DNA base pair editing without double-strand breaks.
Cas12: Distinct cutting mechanism and target recognition; gene editing with sticky ends smaller size and diagnostic applications.
Cas13: RNA targeting CRISPR system; RNA editing, RNA-based therapies, virus targeting.
CRISPR for Epigenetics: Modulates gene depression without altering DNA sequence; epigenetic regulation.
Multiplex CRISPR: Simultaneously targeting multiple genes; high-throughput screening.
In Vivo CRISPR: Delivery of CRISPR-Cas9 in living organisms; genome editing in vivo for therapeutic applications.
Molecular Biology Techniques
PCR: Target DNA sequence amplification.
Gel Electrophoresis: DNA fragment separation by size.
DNA Sequencing: Determining the nucleotide sequence of a DNA molecule.
DNA Fingerprinting: Individual identification through DNA analysis
How Probes Locate DNA Fragments
Probes are designed to bind with the complementary sequence during nucleic acid hybridization within the sample
PCR Requirements
*DNA template
*Primers
*DNA polymerase
*Nucleotides
*Buffer solution
*Magnesium ions
*Thermocycler
*Optional components
Pcr Steps
Denaturation
Annealing
Extension
Amplification cycles
Final extension (optional)
Cooling (optional)
Applications
*DIagnostic
*Forensic science
*Medicine, agricultural, etc
Limitations
*Primer desing, can lead to non-sepcific amplification
*DNA quality and quantity - if poor= results are not reliable
*Amplification bias -some DNA regions cannot be amplified well due to GC content
*High error rate since PCR lacks proofreading
Limited ability to detect low abundance targets
Plasmid Cloning Vectors requirement:
Origin of replication
Selectable marker gene
Multiple cloinig sites (MCS)
Selecting cells for transformation:
use of antibiotics, or blue screening