objectives combo

Eukaryotic DNA Replication

• Comparison with Prokaryotic Replication:

  • Eukaryotic DNA replication is more complex than prokaryotic replication.
  • Eukaryotic DNA synthesis rate is slower.
  • Okazaki fragments are shorter in eukaryotes.
  • Both processes involve major proteins, but they differ in type and function.

• Evolutionary Complexity and Origins of Replication:

  • There's a proposed relationship between the evolutionary complexity of eukaryotes and the number of origins of replication.

• DNA Replication Licensing:

  • DNA replication licensing in eukaryotic cells ensures that replication occurs only once per cell cycle.

• Initiation in S Phase:

  • Eukaryotic DNA replication initiates during the S phase of the cell cycle.

• Major Steps and Key Proteins:

  • Key proteins include Mcm2-7.
  • Mcm2-7 functions relate to prokaryotic DNA replication counterparts.

• Eukaryotic DNA Polymerases:

  • Eukaryotes have several DNA polymerases with distinct features.

• Pol α Function:

  • Pol α functions as a primase.
  • It possesses both primase and DNA polymerase activity.
  • It adds both RNA and DNA primers.

• RNA Primer Removal:

  • RNA primers are removed by RNase H1 and FEN1/RTH1.

• Chromatin and Nucleosome Effects:

  • The presence of chromatin and nucleosomes affects DNA replication.

• Fidelity of DNA Replication:

  • Eukaryotes have mechanisms to ensure high fidelity of DNA replication.

• Mismatch Repair Assay:

  • A biochemical assay with a mismatch plasmid can study nick-directed mismatch repair.
  • The mismatch sequence can be identified, and the repaired sequence predicted.
  • Results after restriction enzyme cleavage can also be predicted.

Telomeres and Telomerase

• Definitions:

  • G-rich strand: Strand rich in guanine.
  • C-rich strand: Strand rich in cytosine.
  • Telomere: Direct repeating sequence at the end of a chromosome.
  • Telomerase: Enzyme that adds telomeric repeats.
  • T-loop: A loop structure formed by the telomere.
  • D-loop: A displacement loop within the telomere.
  • G-quartet: A structure formed by guanine bases.
  • Telomere erosion: Shortening of telomeres over time.
  • Hayflick limit: The number of divisions a normal cell can undergo before senescence.
  • Crisis: A state where cells with critically short telomeres undergo apoptosis or become cancerous.

• Structural Feature of Chromosome End:

  • Linear chromosome ends are capped with telomeres.
  • Telomeres have a G-rich repeating sequence with a single-strand overhang.

• Telomere Protection:

  • Telomeric ends are protected by T-loops, D-loops, G-quartets, and proteins binding to single-stranded and double-stranded regions of the telomere.

• Dicentric Chromosome Formation:

  • Dicentric chromosomes can form when telomeres are lost or damaged.

• Telomere Detection Techniques:

  • Terminal Restriction Fragment (TRF) coupled genomic Southern blot: Measures average telomere length by TRF and Interpret TRF results.
  • Fluorescence In Situ Hybridization (FISH): Relates FISH signal intensity to telomere length.

• Probes:

  • Both TRF and FISH experiments use probes to detect telomeres.

• Shelterin:

  • Shelterin is a protein complex that protects telomeres.

• Retinoblastoma Protein (RB) and p53:

  • RB and p53 are central tumor suppressors.

• Telomere Shortening Effects:

  • Chromosome changes, genome instability, and effects on cell division occur when telomere shortening reaches critical stages (M1, M2) in normal somatic cells.

• Stable Telomere Length:

  • Germ cells and stem cells have stable telomere lengths.

• Cancer Cells and Crisis:

  • Cancer cells bypass crisis and grow indefinitely.

• Telomerase Discovery:

  • The experimental approach used Tetrahymena as a model organism.
  • Treatment was used to confirm the protein and RNA components of telomerase.

• Telomerase Assay Components:

  • Essential components are required for the reaction and interpret the telomerase assay gel result.
  • No DNA template is needed in the assay.
  • The result shows a laddering banding pattern.
  • Telomerase generates laddering bands by adding telomeric repeats.

• RNA Component as Template:

  • Specific experiments and main evidence proved that the RNA component of telomerase serves as a template for telomeres in Tetrahymena.

• TERT Definition:

  • TERT is telomerase reverse transcriptase.

• hTERT Gene Introduction:

  • Introducing the hTERT gene in mortal cells affects telomere length and cell proliferation.

E. coli DNA Replication

• OriC Structural Features:

  • Structural features of OriC enable it to start replication and prevent accidental replication.
  • A/T-rich regions have biochemical significance.

• Role of DnaA:

  • DnaA plays a role in DNA replication.
  • DnaA mediates the initiation of DNA replication and creates the replication bubble at OriC.

• Role of DnaB and DnaC:

  • DnaB and DnaC play a role in DNA replication.
  • Release of DnaC marks the initiation of the replication fork moving away from OriC.

• Elongation Phase Proteins:

  • Various proteins play a role in the elongation phase, including DnaB, topoisomerase, single-strand binding protein, DnaG primase, sliding clamp, clamp loader, and DNA polymerase.

• E. coli DNA Polymerases (I to V):

  • Discovery of DNA pol I involved fractionation and in vitro assay.
  • DNA pol I has polymerase, 3’-5’ exonuclease, and 5’-3’ exonuclease activities.
  • Key evidence supports DNA pol III as the main replicative polymerase.

• Definitions:

  • Klenow fragment: A fragment of DNA polymerase I.
  • 3’-5’ exonuclease: An exonuclease that removes nucleotides from the 3' end of a DNA strand.
  • 5’-3’ exonuclease: An exonuclease that removes nucleotides from the 5' end of a DNA strand.

• DNA Polymerase I Reaction Mechanism:

  • The reaction mechanism relates to the structure features (finger, palm, and thumb) of DNA pol I.
  • $Mg^{2+}$ ions are needed in the active site for each nucleotide addition; two $Mg^{2+}$ ions are needed in the active site for each run of nucleotide addition.
  • $Mg^{2+}$ ions play a role in the catalysis of nucleotide addition.

• E. coli DNA Polymerase III Holoenzyme:

  • The holoenzyme has four subassemblies.
  • The core polymerase composition is specific.
  • Different components coordinate leading and lagging strand DNA synthesis.

• Definitions:

  • Processivity: The ability of an enzyme to catalyze consecutive reactions without releasing its substrate.
  • Processive enzyme: An enzyme with high processivity.
  • Distributive enzyme: An enzyme with low processivity.
  • De novo synthesis: Synthesis from scratch.
  • Non-de novo synthesis: Synthesis that requires a primer or template.

• Processivity and DNA Synthesis Rate:

  • High processivity relates to the rate of DNA synthesis in E. coli.

• DNA Primase Functional Features:

  • De novo synthesis, error-prone, and distributive.

• RNaseH Definition:

  • RNaseH is an enzyme that degrades RNA in RNA-DNA hybrids.

• RNA Primer Removal and Ligation:

  • Proteins are required for removing RNA primers and ligating Okazaki fragments.
  • Two classes of ligases exist with corresponding cofactors.

• DNA Ligase Reaction Mechanism:

  • The reaction mechanism of DNA ligase is specific.

• Termination of E. coli Replication:

  • Tus and ter: Tus binds to ter sites to arrest DnaB helicase.
  • Asymmetric binding traps/arrests DnaB helicase and the replication fork in a directional manner.
  • Permissive vs. non-permissive directions exist for the two DNA replication forks.

• Fidelity of DNA Replication:

  • DNA synthesis has high fidelity due to three steps.
  • Purine-purine or pyrimidine-pyrimidine pairs inside the double helix pose a structural challenge.

• Forward Genetic Screening:

  • Forward genetic screening identifies genes and components affecting the fidelity of DNA synthesis.
  • Mutant E. coli strains (mutator and anti-mutator) have specific phenotypes and functional changes.

• DNA Polymerase Nucleotide Selectivity:

  • The mechanism of nucleotide selectivity is specific.
  • The finger domain and O-helix mediate nucleotide selectivity.
  • dNTP is coordinated in the active site and added to the 3’OH of the new strand.

• 3’-5’ Exonuclease Proofreading:

  • The mechanism of the second proofreading step is performed by the 3’-5’ exonuclease of DNA polymerase.

• Strand-Directed Mismatch Repair:

  • The mechanism of strand-directed mismatch repair in E. coli is specific.
  • Steps include identifying the repaired strand and the role of methylation.

• Mismatch Repair Assay:

  • A biochemical assay with a mismatch plasmid can study methylation strand-directed mismatch repair.
  • The mismatch sequence can be identified, and the repaired sequence predicted.
  • Results after restriction enzyme cleavage can also be predicted.

Molecular Techniques (Part II)

• PCR Principle:

  • PCR involves denaturing, annealing, and elongation steps.
  • PCR amplification curve includes different phases.

• $T_m$ Calculation:

  • $T_m$ for primers can be calculated and used to estimate the annealing temperature.

• PCR Applications:

  • PCR has various applications.
  • Plateau vs. exponential phase.

• Real-Time PCR Principle:

  • Real-time PCR uses indirect detection (or non-specific detection) of PCR production by SYBR green.
  • DNA structures affect SYBR green fluorescence.

• Definitions:

  • Threshold: The level of fluorescence above background.
  • $C_t$ value: The cycle at which fluorescence crosses the threshold.

• Quantitative PCR:

  • $C_t$ value quantifies target DNA using a standard curve.
  • $ΔC<em>t$ value quantifies the molar ratio of two samples using the equation $(2^{ΔC</em>t})$.

• Taqman Probes:

  • Taqman probes enable sequence-specific detection of DNA synthesis in real-time PCR.
  • Multiplex PCR can be performed using Taqman real-time PCR.

• Microarray Principle:

  • Microarrays use temperature and salt conditions to increase wash stringency.
  • Spotted/printed material on the microarray spots allows detection of gene expression.

• cDNA Microarray Interpretation:

  • cDNA microarray results determine relative expression levels.

• RNA-Sequencing Principle:

  • RNA-sequencing involves general procedures.
  • Oligo-dT beads purify mRNA.

• RNA-Sequencing Advantage:

  • RNA-sequencing has advantages over microarray analysis.

Genome Organization and Prokaryotic Genome Packaging

• Definitions:

  • Genome: The total genetic material of an organism.
  • Nucleoid: The irregularly-shaped region within a prokaryotic cell where the genetic material is localized.
  • C-value: The amount of DNA in a haploid genome.
  • Gene density: The number of genes per unit length of DNA.

• DNA Amount vs. C-Value:

  • DNA amount and C-value differ.
  • Ploidy affects the DNA amount.

• Gene Number and Eukaryotic Complexity:

  • The number of genes links to the complexity of eukaryotic organisms.

• Definitions:

  • $C_0t$ value: The initial concentration of DNA multiplied by the time of incubation.

• $C_0t$ Analysis Principle:

  • $C<em>0t$ analysis explains how the $C</em>0t$ value is determined.

• Genome Size and $C_0t$ Value:

  • The size of the genome affects the $C_0t$ value.

• Repetitive DNA and $C_0t$ Value:

  • The amount of repetitive DNA affects the $C<em>0t$ value (low $C</em>0t$ - highly repetitive; middle $C<em>0t$ - middle repetitive; high $C</em>0t$ – single copy).

• Definitions:

  • Satellite DNA: Highly repetitive DNA found in centromeres and telomeres.
  • Mini-satellite DNA: Repetitive DNA with repeat units of 10-100 base pairs.
  • Micro-satellite DNA: Repetitive DNA with short repeat units of 1-6 base pairs.
  • Transposable elements: DNA sequences that can change their position within a genome.
  • LINEs: Long interspersed nuclear elements.
  • SINEs: Short interspersed nuclear elements.
  • Alu elements: A type of SINE.

• High Repetitive DNA:

  • Two types are simple repetitive (tandem repeats) and complex repeat (interspersed elements).

• Satellite DNA Detection:

  • Satellite, mini-satellite, and micro-satellite DNA can be detected by CsCl gradient centrifugation.

• Repetitive Sequence Amount:

  • The relative amount of total repetitive sequence vs. unique sequence in the human genome and on single chromosomes is specific.

• Middle Repetitive DNA:

  • Middle repetitive DNA exists (e.g., rDNA).
  • rDNA gene localization and transcription resemble “Christmas trees”.

• Single-Copy DNA Percentage:

  • The percentage of single-copy DNA and protein-coding genes in the human genome is specific.

• C-Value Paradox:

  • The C-value paradox and its factors are specific.
  • Genome complexity is measured by the proteome, not the number of genes.

• E. coli Genome Packaging:

  • Proteins help E. coli DNA generate and maintain supercoiling and compact dimensions.

• DNA Length Calculation:

  • If the genome size is known (e.g., human genome has 6.6 billion bp of DNA), the total length of B-form DNA can be calculated.

• Definitions:

  • Histones: Proteins around which DNA wraps to form nucleosomes.
  • Core histones: H2A, H2B, H3, and H4.
  • Linker histones: H1.
  • Nucleosomes: The basic units of chromatin structure.
  • Octamer: The core histone complex (H2A, H2B, H3, H4)2.
  • Chromatin: The complex of DNA and proteins that makes up chromosomes.

• Histone Fold/Hand-Shake Motif:

  • The histone fold/hand-shake motif contributes to nucleosome structure assembly.
  • The nucleosome composition differs in transcriptionally active vs. transcriptionally silenced regions.
  • DNA wraps around histones in a nucleosome.
  • Proteins are required for nucleosome assembly.

Molecular Cloning and Useful Enzymes

• Useful Enzymes:

  • Useful enzymes are phosphatase, nuclease, endonuclease, and endonuclease.
  • Application of each enzyme.

• Definitions:

  • Restriction enzyme isoschizomer
  • Neoschizomer

• Restriction Enzymes and Palindromes:

  • Restriction enzymes use palindromes to recognize specific sequences and generate different sticky or blunt ends.
  • Different ends (3’-overhang, 5’-overhang, and blunt end) and compatible ends can be recognized.

• Enzymes for Cloning:

  • Different enzymes (restriction enzyme, DNA ligase, phosphatase, and kinase) generate different compatible DNA fragment ends for cloning.
  • When to apply phosphatase to remove 5’-phosphate to prevent vector re-ligation.
  • T4 kinase to add phosphate to 5’-end.
  • DNA ligase to ligate compatible ends.

• Cloning Tricks:

  • (1) Phosphatase treatment of the vector prevents re-ligation.
  • (2) Blue-white screening selects for recombinant plasmids.
  • (3) DNA polymerase fills in 5’ overhang, and Klenow chews back 3’ overhangs to produce compatible blunt ends.
  • (4) Linkers or adaptors are added to produce known sequence or restrict site ends.

• Clone Testing:

  • Two ways to test if a clone contains the desired insert: restriction mapping and sequencing.

DNA Sequencing

• Sanger Sequencing Principle:

  • Role of ddNTP in the Sanger sequencing reaction.
  • Sequence results are interpreted to derive synthesized DNA fragments and template DNA sequences.

• Pyrosequencing Principle:

  • Stoichiometric release of pyrophosphate and sequential addition of one dNTP at a time are used in pyrosequencing.
  • Roles of sulfurylase, luciferase, and apyrase.
  • Sequence results are interpreted to derive synthesized DNA fragments and template DNA sequences.

• Reversible Terminator Sequencing Principle:

  • Fluorescent reversible terminator dNTPs are used in reversible terminator sequencing.

Genome Organization and Prokaryotic Genome Package

• Definitions:

  • Genome:
  • Nucleoid:
  • C-value:
  • Gene density
  • DNA amount and C-value differ.
  • Ploidy affects the DNA amount.

• Gene Number and Eukaryotic Complexity:

  • The number of genes links to the complexity of eukaryotic organisms.

• Definitions:

  • $C_0t$ value

• $C_0t$ Analysis Principle:

  • Explains how the $C_0t$ value is determined.

• Genome Size and $C_0t$ Value:

  • The size of the genome affects the $C_0t$ value.

• Repetitive DNA and $C_0t$ Value:

  • The amount of repetitive DNA affects the $C<em>0t$ value (low $C</em>0t$ - highly repetitive; middle $C<em>0t$ - middle repetitive; high $C</em>0t$ – single copy).

• Definitions:

  • Satellite DNA:
  • Mini-satellite DNA:
  • Micro-satellite DNA:
  • Transposable elements:
  • LINEs:
  • SINEs:
  • Alu elements:

• High Repetitive DNA:

  • Two types: simple repetitive (tandem repeats) and complex repeat (interspersed elements).

• Satellite DNA Detection:

  • Satellite, mini-satellite, and micro-satellite DNA can be detected by CsCl gradient centrifugation.

• Repetitive Sequence Amount:

  • Location and general percentage of total repetitive sequence in the human genome and on single chromosomes.

• Middle Repetitive DNA:

  • Middle repetitive DNA exists (e.g., rDNA).
  • rDNA gene localization and transcription resemble “Christmas trees”.

• Single-Copy DNA Percentage:

  • The percentage of single-copy DNA and protein-coding genes in the human genome.

• C-Value Paradox:

  • The C-value paradox and its factors.
  • Genome complexity is measured by the proteome, not the number of genes.

• E. coli Genome Packaging:

  • Proteins help E. coli DNA generate and maintain supercoiling and compact dimensions.

Eukaryotic Genome Organization and DNase I Sensitivity Mapping

• DNA Length Calculation:

  • If the genome size is known (e.g., human genome has 6.6 billion bp of DNA), the total length of B-form DNA can be calculated.

• Definitions:

  • Histones:
  • Core histones:
  • Linker histones:
  • Nucleosomes:
  • Octamer:
  • Chromatin:

• Histone Fold/Hand-Shake Motif:

  • The histone fold/hand-shake motif contributes to nucleosome structure assembly.
  • The nucleosome composition differs in transcriptionally active vs. transcriptionally silenced regions.
  • DNA wraps around histones in a nucleosome.
  • Proteins are required for nucleosome assembly.

• Chromosome Structure:

  • p and q arm, centromere: Defined.
  • Types of metaphase chromosomes and position of the centromere relative to p and q arm.

• Human Chromosome Nomenclature:

  • Related to DNA amount/content of the chromosome.

• Definitions:

  • Cytogenetics
  • Aneuploidy
  • Sister-chromatids
  • Karyotype

• Karyotype Interpretation:

  • Ability to interpret a karyotype.

• SKY Principle:

  • A common application of SKY is described.
  • The key reagent-chromosome-specific probe is generated.
  • The role of Cot DNA in generating a chromosome-specific probe; what happens if Cot DNA is missing (nonspecific probe binding, colors?).
  • How does the repetitive sequence affect the SKY technique.

• Euchromatin Properties:

  • Related to active genes.

• Definitions:

  • Nuclease:
  • MNase:
  • DNase:

• Weintraub’s Experiment (1980):

  • Hypothesis, experimental design, observation, and conclusion of the experiment are described.

• Euchromatin vs. Heterochromatin:

  • Comparison and contrast.

• Constitutive vs. Facultative Heterochromatin:

  • Comparison and contrast.

• DNase I Sensitivity vs. DNase I Hypersensitivity:

  • Comparison and contrast.

• Genomic Southern Blot Analysis:

  • Principle of genomic Southern blot analysis to map DNase I HSS.

• Chromatin State and Transcription:

  • Nuclear sensitivity and DNase I HSS relate to chromatin state (open vs. condensed chromatin) and transcription (active gene vs. inactive gene) using LCR of β-globin gene locus as an example.

• Hispanic Deletion Patients:

  • What is genetically deleted in Hispanic deletion patients.
  • How genetic alteration leads to thalassemia.

• LCR of β-Globin Gene Locus:

  • Role of LCR of β-globin gene locus and predicted impact of its deletion and mutation on β-globin gene locus (chromatin state, gene expression, replication timing, and developmental regulation).

• LCR and Position Effect:

  • LCR's role is related to the position effect and copy number-dependent expression in β-globin gene transgenic mice.

• LCR HS-2:

  • Structural features and regulatory elements of LCR HS-2 and its role in transcription regulation, related to DNase sensitivity, nucleosome depletion state, and transcriptional factor binding sites of this region.

• Long-Range Regulatory Elements and Cis-Regulatory Elements:

  • Related to DNase I sensitivity and histone/nucleosome density.

• DNase I-Seq Principle:

  • Information obtained through genome-wide DNase I-Seq and significance of DNase I-Seq are described.

• Biotin:

  • Biotin definition.

• Biotin-Avidin Interaction:

  • Application in DNase I-Seq is described.

Week 1 Discovery of DNA as the Molecule of Inheritance

• Central Dogma:

  • The central dogma of molecular biology.

• Definitions:

  • Genes:
  • Genotype:
  • Phenotype:
  • Genetics:

• Paul Berg’s Experiment (1971):

  • Experimental design and significance of the experiment are described.

• Mendel’s Laws of Inheritance:

  • Description of Mendel’s Laws.

• Miescher’s Experiment:

  • The first to “isolate” “nuclein”.
  • Procedures and principles behind DNA purification.
  • The chemical composition of nucleic acid discovered by Miescher’s research.

• Criteria for Inheritance Molecule:

  • Reasons why DNA wasn't considered the carrier of genetic information in Miescher’s time (emphasize historic – 20 aa proteins, more combinations).

• Fleming’s Findings:

  • How findings about chromatin/chromosome contribute to the discovery of DNA as the molecule of inheritance.

• Thomas Morgan’s Research:

  • Research approaches and findings.
  • How Morgan’s discovery contradicts Mendel’s first law.

• Hermann Muller’s Research:

  • Research approaches and findings.
  • How Muller’s research contributes to the discovery of DNA as the molecule of inheritance.

• Beadle and Tatum’s Experiment:

  • Choice of model organism, hypothesis, experimental design, and observation/conclusion interpretation.
  • Significance of Beadle and Tatum’s research.
  • Why model organisms are used in scientific experiments.

• Forward vs Reverse Genetic Screen:

  • Comparison and contrast.
  • Why Beadle and Tatum’s experiment is a type of forward genetic screen.

• One Gene: One Enzyme Hypothesis:

  • Definition and an example of an exception.

• Griffith’s Experiment:

  • Observation and conclusion interpretation.
  • Definition of transformation and description of the molecular basis and principle of transformation.
  • Significance of Griffith’s research.

• Avery, Macleod, and McCarty’s Experiment:

  • Hypothesis, experimental design, and observation/conclusion interpretation.
  • Significance of Avery, Macleod, and McCarty’s research.

• Hershey and Chase’s Blender Experiment:

  • Hypothesis, experimental design, and observation/conclusion interpretation.
  • Significance of Hershey and Chase’s research; why it is referred to as a blender experiment.

• DNA as Molecule of Inheritance:

  • How research from Avery et al. and Hershey et al. demonstrated that DNA is the molecule of inheritance.

Week 2 DNA Structure and Chemical Properties

• DNA Structure:

  • Description of DNA structure using appropriate terminology.

• DNA Polarity:

  • Explanation of DNA polarity.

• DNA and RNA Chemical Structure:

  • Review of the chemical structure of DNA and RNA components.
  • Chemical structure of the nitrogenous bases in DNA and RNA.
  • Definitions of purines and pyrimidines.

• Spontaneous Deamination:

  • Description of spontaneous deamination of 5mC and C.
  • How 5mC deamination relates to spontaneous mutagenesis and the steps from 5mC deamination to permanent mutation in the DNA genome.

• Covalent Bond and Nucleoside Conformation:

  • Name of the covalent bond between ribose and a nitrogenous base.
  • Difference between syn- and anti- conformation found in nucleosides.
  • How the conformation of nucleosides relates to different forms of DNA.

• Apurinic Sites:

  • Definition of apurinic sites.

• Chargaff’s Research:

  • Research approaches and findings.
  • How Chargaff’s research rejects the tetranucleotide hypothesis.

• Rosalind Franklin’s Research:

  • Research approaches and findings.
  • How the X-ray diffraction pattern relates to the A form and B form DNA in Franklin’s experiment and how A form and B form DNA were obtained for her experiments.

• DNA Models:

  • Comparison and contrast of Linus Pauling’s DNA model and the DNA model by Watson and Crick.
  • Why Linus Pauling’s DNA model is unstable and incorrect.

• Known Facts Before DNA Structure:

  • Known facts about DNA before DNA structure was known.
  • How this knowledge helps build a DNA 3D model (base-pairing, anti-parallel, B-form double helix).

• DNA Structure Properties:

  • Basic properties of DNA structure: diameter, distance between adjacent two stacking bases, distance per turn, major groove and minor groove, #bp per helical turn.

• DNA Helix Forms:

  • Comparison and contrast of the structural properties of the three forms of helices (A, B, and Z form).
  • Significance and effect of each form of helix on DNA and RNA structure.
  • Nucleotide conformation, handedness, and structural features of the Z-form helices and the organisms that contain Z-form DNA.

• Forces Stabilizing DNA Structure:

  • Forces that stabilize DNA structure (base-pairing, base stacking, and phosphate backbone).

• Factors Affecting DNA Stability:

  • Roles of different factors that affect DNA stability (heat, pH, salt concentration).

• DNA Denaturation and Renaturation:

  • Explanation and prediction of DNA denaturation and renaturation under different conditions.
  • Determination of Tm based on the nucleotide sequence using short equation (T_m = 4 \times # of (G+C) + 2 \times # of (A+T)).
  • Determination of DNA concentrations based on UV absorption reading.

Week 2 RNA Structure and Chemical Properties

• DNA vs RNA Structure:

  • Structure difference between DNA and RNA.
  • The role of 2’-OH in forming RNA structure.

• RNA Secondary and Tertiary Structure:

  • Molecular basis of RNA secondary and tertiary structure.
  • The role of RNA 2’-OH in forming A-form short helices as well as the role of wobble base-pairing in forming RNA secondary and tertiary structure.

• RNA Hydrolysis:

  • The molecular basis of RNA hydrolysis in an alkaline solution.

• Catalytic RNA:

  • The molecular basis of catalytic RNA-mediated reactions (RNaseP mediated RNA cleavage and hammerhead RNA cleavage).
  • The role of $Mg^{2+}$ ions in RNA-mediated catalysis.

• RNA Types and Distribution:

  • Types of different RNA and their distribution in the cell.

• lncRNA:

  • The role of lncRNA using Xist as an example.

DNA Topology

• DNA Topological Problems:

  • DNA topological problems during DNA replication.

• Linking Number, Twist, and Writhe:

  • Defining Lk, Lk0, ΔLk, Tw, and Wr and Lk formula. Use $(Lk=Tw+Wr)$ to calculate Lk, Tw, and Wr in B-form cccDNA.

• Supercoils:

  • Two types of writhe supercoils, interwound or plectonemic vs toroid or spiral.
  • Handedness of different supercoils in cccDNA and nucleosome DNA.

• Supercoils in Organisms:

  • Different types of supercoils found in bacteria, eukaryotes, and some thermophiles and their biological significance.

• Effects on DNA Topology:

  • Use $(Lk=Tw+Wr)$ to predict the change of Lk, Tw, and Wr under different conditions (topoisomerases, EtBr, wrap around histones, etc.).

• DNase I Effect:

  • Effect of DNase I on DNA topology, on Lk, Tw, and Wr.

• EtBr Effect:

  • Effect of EtBr on DNA topology, on Lk, Tw, and Wr.

• **