Nucleic Acids Biochemistry

  1. Tools and Techniques in Nucleic Acid Research
    1. Isolation and Purification of Nucleic Acids
      1. DNA Extraction Methods
        1. Cell Lysis Techniques
          1. Protein Removal
            1. Organic Extraction
              1. Column Purification
              2. RNA Extraction Methods
                1. RNase Inhibition
                  1. Guanidinium-Based Methods
                    1. Phenol-Chloroform Extraction
                      1. Silica-Based Purification
                      2. Removal of Contaminants
                        1. Protein Contamination
                          1. RNA Contamination
                            1. Salt Removal
                              1. Organic Solvent Removal
                              2. Quality Assessment
                                1. Integrity Analysis
                                  1. Purity Measurements
                                    1. Concentration Determination
                                  2. Quantification of Nucleic Acids
                                    1. UV Spectrophotometry
                                      1. Absorption at 260 nm
                                        1. A260/A280 Ratio
                                          1. A260/A230 Ratio
                                            1. Extinction Coefficients
                                            2. Fluorescence-Based Methods
                                              1. Intercalating Dyes
                                                1. Minor Groove Binders
                                                  1. Sequence-Specific Probes
                                                    1. Real-Time Detection
                                                    2. Other Quantification Methods
                                                      1. Diphenylamine Assay
                                                        1. Orcinol Assay
                                                          1. Picogreen Assay
                                                        2. Gel Electrophoresis
                                                          1. Principles of Electrophoresis
                                                            1. Charge-to-Mass Ratio
                                                              1. Electric Field Effects
                                                                1. Migration Patterns
                                                                  1. Resolution Factors
                                                                  2. Agarose Gel Electrophoresis
                                                                    1. Gel Preparation
                                                                      1. DNA Separation
                                                                        1. Size-Based Separation
                                                                          1. Linear vs Supercoiled DNA
                                                                            1. Molecular Weight Markers
                                                                            2. Visualization Techniques
                                                                              1. Ethidium Bromide Staining
                                                                                1. SYBR Safe Staining
                                                                                  1. Methylene Blue Staining
                                                                                    1. UV Transillumination
                                                                                  2. Polyacrylamide Gel Electrophoresis
                                                                                    1. Gel Composition
                                                                                      1. DNA and RNA Separation
                                                                                        1. High Resolution Separation
                                                                                          1. Single Nucleotide Resolution
                                                                                            1. Sequencing Gels
                                                                                            2. Denaturing vs Native Gels
                                                                                              1. Urea-PAGE
                                                                                                1. Formamide Gels
                                                                                                  1. Native PAGE Applications
                                                                                                2. Pulsed-Field Gel Electrophoresis
                                                                                                  1. Large DNA Separation
                                                                                                    1. Chromosome Analysis
                                                                                                      1. Bacterial Typing
                                                                                                    2. Nucleic Acid Hybridization
                                                                                                      1. Principles of Hybridization
                                                                                                        1. Complementary Base Pairing
                                                                                                          1. Kinetics of Hybridization
                                                                                                            1. Stringency Conditions
                                                                                                              1. Melting Temperature
                                                                                                              2. Probe Design and Labeling
                                                                                                                1. Probe Length Considerations
                                                                                                                  1. GC Content Effects
                                                                                                                    1. Secondary Structure Avoidance
                                                                                                                      1. Labeling Methods
                                                                                                                        1. Radioactive Labels
                                                                                                                          1. Fluorescent Labels
                                                                                                                            1. Enzymatic Labels
                                                                                                                              1. Chemiluminescent Labels
                                                                                                                            2. Southern Blotting
                                                                                                                              1. DNA Transfer Methods
                                                                                                                                1. Capillary Transfer
                                                                                                                                  1. Vacuum Transfer
                                                                                                                                    1. Electroblotting
                                                                                                                                    2. Membrane Types
                                                                                                                                      1. Nitrocellulose
                                                                                                                                        1. Nylon Membranes
                                                                                                                                        2. Detection Methods
                                                                                                                                          1. Autoradiography
                                                                                                                                            1. Colorimetric Detection
                                                                                                                                              1. Chemiluminescence
                                                                                                                                            2. Northern Blotting
                                                                                                                                              1. RNA Transfer Considerations
                                                                                                                                                1. Denaturing Conditions
                                                                                                                                                  1. RNase Precautions
                                                                                                                                                    1. Size Determination
                                                                                                                                                    2. In Situ Hybridization
                                                                                                                                                      1. Tissue Preparation
                                                                                                                                                        1. Probe Penetration
                                                                                                                                                          1. Signal Detection
                                                                                                                                                            1. Fluorescence In Situ Hybridization
                                                                                                                                                          2. Polymerase Chain Reaction
                                                                                                                                                            1. Principles and Components
                                                                                                                                                              1. Template DNA
                                                                                                                                                                1. Primer Design
                                                                                                                                                                  1. DNA Polymerase Selection
                                                                                                                                                                    1. Buffer Composition
                                                                                                                                                                      1. Magnesium Concentration
                                                                                                                                                                      2. Steps of PCR
                                                                                                                                                                        1. Denaturation
                                                                                                                                                                          1. Temperature Requirements
                                                                                                                                                                            1. Time Considerations
                                                                                                                                                                              1. DNA Melting
                                                                                                                                                                              2. Annealing
                                                                                                                                                                                1. Primer Binding
                                                                                                                                                                                  1. Temperature Optimization
                                                                                                                                                                                    1. Specificity Control
                                                                                                                                                                                    2. Extension
                                                                                                                                                                                      1. Polymerase Activity
                                                                                                                                                                                        1. Extension Time
                                                                                                                                                                                          1. Product Synthesis
                                                                                                                                                                                        2. Primer Design Principles
                                                                                                                                                                                          1. Length Considerations
                                                                                                                                                                                            1. GC Content
                                                                                                                                                                                              1. Melting Temperature
                                                                                                                                                                                                1. Specificity Requirements
                                                                                                                                                                                                  1. Primer Dimers Avoidance
                                                                                                                                                                                                  2. Variants of PCR
                                                                                                                                                                                                    1. Reverse Transcription PCR
                                                                                                                                                                                                      1. RNA Template
                                                                                                                                                                                                        1. Reverse Transcriptase
                                                                                                                                                                                                          1. One-Step vs Two-Step
                                                                                                                                                                                                          2. Quantitative PCR
                                                                                                                                                                                                            1. Real-Time Detection
                                                                                                                                                                                                              1. Fluorescent Reporters
                                                                                                                                                                                                                1. Quantification Methods
                                                                                                                                                                                                                  1. Standard Curves
                                                                                                                                                                                                                  2. Multiplex PCR
                                                                                                                                                                                                                    1. Multiple Target Amplification
                                                                                                                                                                                                                      1. Primer Compatibility
                                                                                                                                                                                                                        1. Product Discrimination
                                                                                                                                                                                                                        2. Nested PCR
                                                                                                                                                                                                                          1. Increased Specificity
                                                                                                                                                                                                                            1. Two-Round Amplification
                                                                                                                                                                                                                              1. Contamination Prevention
                                                                                                                                                                                                                              2. Long-Range PCR
                                                                                                                                                                                                                                1. Large Product Amplification
                                                                                                                                                                                                                                  1. High-Fidelity Polymerases
                                                                                                                                                                                                                                    1. Optimization Strategies
                                                                                                                                                                                                                                2. DNA Sequencing
                                                                                                                                                                                                                                  1. Sanger Sequencing
                                                                                                                                                                                                                                    1. Chain Termination Method
                                                                                                                                                                                                                                      1. Dideoxynucleotides
                                                                                                                                                                                                                                        1. DNA Polymerase Reaction
                                                                                                                                                                                                                                          1. Termination Events
                                                                                                                                                                                                                                          2. Reaction Components
                                                                                                                                                                                                                                            1. Template DNA
                                                                                                                                                                                                                                              1. Sequencing Primers
                                                                                                                                                                                                                                                1. DNA Polymerase
                                                                                                                                                                                                                                                  1. dNTPs and ddNTPs
                                                                                                                                                                                                                                                  2. Detection Methods
                                                                                                                                                                                                                                                    1. Radioactive Labeling
                                                                                                                                                                                                                                                      1. Fluorescent Labeling
                                                                                                                                                                                                                                                        1. Capillary Electrophoresis
                                                                                                                                                                                                                                                        2. Data Analysis
                                                                                                                                                                                                                                                          1. Chromatogram Interpretation
                                                                                                                                                                                                                                                            1. Base Calling
                                                                                                                                                                                                                                                              1. Quality Scores
                                                                                                                                                                                                                                                            2. Next-Generation Sequencing
                                                                                                                                                                                                                                                              1. Principles and Platforms
                                                                                                                                                                                                                                                                1. Illumina Sequencing
                                                                                                                                                                                                                                                                  1. Ion Torrent Sequencing
                                                                                                                                                                                                                                                                    1. PacBio Sequencing
                                                                                                                                                                                                                                                                      1. Oxford Nanopore Sequencing
                                                                                                                                                                                                                                                                      2. Library Preparation
                                                                                                                                                                                                                                                                        1. DNA Fragmentation
                                                                                                                                                                                                                                                                          1. Adapter Ligation
                                                                                                                                                                                                                                                                            1. Size Selection
                                                                                                                                                                                                                                                                              1. Amplification
                                                                                                                                                                                                                                                                              2. Sequencing Chemistry
                                                                                                                                                                                                                                                                                1. Sequencing by Synthesis
                                                                                                                                                                                                                                                                                  1. Sequencing by Ligation
                                                                                                                                                                                                                                                                                    1. Single-Molecule Sequencing
                                                                                                                                                                                                                                                                                    2. Data Output and Analysis
                                                                                                                                                                                                                                                                                      1. Read Length
                                                                                                                                                                                                                                                                                        1. Coverage Depth
                                                                                                                                                                                                                                                                                          1. Error Rates
                                                                                                                                                                                                                                                                                            1. Bioinformatics Processing
                                                                                                                                                                                                                                                                                        2. Recombinant DNA Technology
                                                                                                                                                                                                                                                                                          1. Restriction Enzymes
                                                                                                                                                                                                                                                                                            1. Types and Classification
                                                                                                                                                                                                                                                                                              1. Type I Enzymes
                                                                                                                                                                                                                                                                                                1. Type II Enzymes
                                                                                                                                                                                                                                                                                                  1. Type III Enzymes
                                                                                                                                                                                                                                                                                                  2. Recognition Sequences
                                                                                                                                                                                                                                                                                                    1. Palindromic Sequences
                                                                                                                                                                                                                                                                                                      1. Cut Site Patterns
                                                                                                                                                                                                                                                                                                        1. Overhang Generation
                                                                                                                                                                                                                                                                                                        2. Specificity and Activity
                                                                                                                                                                                                                                                                                                          1. Star Activity
                                                                                                                                                                                                                                                                                                            1. Buffer Requirements
                                                                                                                                                                                                                                                                                                              1. Temperature Sensitivity
                                                                                                                                                                                                                                                                                                            2. Cloning Vectors
                                                                                                                                                                                                                                                                                                              1. Plasmids
                                                                                                                                                                                                                                                                                                                1. Origin of Replication
                                                                                                                                                                                                                                                                                                                  1. Selectable Markers
                                                                                                                                                                                                                                                                                                                    1. Multiple Cloning Sites
                                                                                                                                                                                                                                                                                                                      1. Copy Number Control
                                                                                                                                                                                                                                                                                                                      2. Bacteriophages
                                                                                                                                                                                                                                                                                                                        1. Lambda Phage Vectors
                                                                                                                                                                                                                                                                                                                          1. M13 Vectors
                                                                                                                                                                                                                                                                                                                            1. Phagemid Vectors
                                                                                                                                                                                                                                                                                                                            2. Cosmids
                                                                                                                                                                                                                                                                                                                              1. Large Insert Capacity
                                                                                                                                                                                                                                                                                                                                1. Packaging Constraints
                                                                                                                                                                                                                                                                                                                                  1. Selection Systems
                                                                                                                                                                                                                                                                                                                                  2. Artificial Chromosomes
                                                                                                                                                                                                                                                                                                                                    1. Bacterial Artificial Chromosomes
                                                                                                                                                                                                                                                                                                                                      1. Yeast Artificial Chromosomes
                                                                                                                                                                                                                                                                                                                                        1. Human Artificial Chromosomes
                                                                                                                                                                                                                                                                                                                                      2. DNA Ligation
                                                                                                                                                                                                                                                                                                                                        1. DNA Ligases
                                                                                                                                                                                                                                                                                                                                          1. T4 DNA Ligase
                                                                                                                                                                                                                                                                                                                                            1. E. coli DNA Ligase
                                                                                                                                                                                                                                                                                                                                              1. Thermostable Ligases
                                                                                                                                                                                                                                                                                                                                              2. Ligation Conditions
                                                                                                                                                                                                                                                                                                                                                1. ATP Requirements
                                                                                                                                                                                                                                                                                                                                                  1. Buffer Composition
                                                                                                                                                                                                                                                                                                                                                    1. Temperature and Time
                                                                                                                                                                                                                                                                                                                                                    2. Blunt-End vs Sticky-End Ligation
                                                                                                                                                                                                                                                                                                                                                      1. Efficiency Differences
                                                                                                                                                                                                                                                                                                                                                        1. Directional Cloning
                                                                                                                                                                                                                                                                                                                                                          1. Adapter Strategies
                                                                                                                                                                                                                                                                                                                                                        2. Transformation and Selection
                                                                                                                                                                                                                                                                                                                                                          1. Competent Cell Preparation
                                                                                                                                                                                                                                                                                                                                                            1. Chemical Competence
                                                                                                                                                                                                                                                                                                                                                              1. Electrocompetence
                                                                                                                                                                                                                                                                                                                                                                1. Natural Competence
                                                                                                                                                                                                                                                                                                                                                                2. Transformation Methods
                                                                                                                                                                                                                                                                                                                                                                  1. Heat Shock
                                                                                                                                                                                                                                                                                                                                                                    1. Electroporation
                                                                                                                                                                                                                                                                                                                                                                      1. Chemical Methods
                                                                                                                                                                                                                                                                                                                                                                      2. Selection Strategies
                                                                                                                                                                                                                                                                                                                                                                        1. Antibiotic Resistance
                                                                                                                                                                                                                                                                                                                                                                          1. Blue-White Screening
                                                                                                                                                                                                                                                                                                                                                                            1. Complementation
                                                                                                                                                                                                                                                                                                                                                                          2. Creation of DNA Libraries
                                                                                                                                                                                                                                                                                                                                                                            1. Genomic Libraries
                                                                                                                                                                                                                                                                                                                                                                              1. Total DNA Representation
                                                                                                                                                                                                                                                                                                                                                                                1. Insert Size Considerations
                                                                                                                                                                                                                                                                                                                                                                                  1. Vector Selection
                                                                                                                                                                                                                                                                                                                                                                                  2. cDNA Libraries
                                                                                                                                                                                                                                                                                                                                                                                    1. mRNA Template
                                                                                                                                                                                                                                                                                                                                                                                      1. Reverse Transcription
                                                                                                                                                                                                                                                                                                                                                                                        1. Expressed Gene Representation
                                                                                                                                                                                                                                                                                                                                                                                        2. Library Construction
                                                                                                                                                                                                                                                                                                                                                                                          1. DNA Preparation
                                                                                                                                                                                                                                                                                                                                                                                            1. Vector Preparation
                                                                                                                                                                                                                                                                                                                                                                                              1. Ligation and Transformation
                                                                                                                                                                                                                                                                                                                                                                                            2. Screening of DNA Libraries
                                                                                                                                                                                                                                                                                                                                                                                              1. Hybridization-Based Screening
                                                                                                                                                                                                                                                                                                                                                                                                1. Colony Hybridization
                                                                                                                                                                                                                                                                                                                                                                                                  1. Plaque Hybridization
                                                                                                                                                                                                                                                                                                                                                                                                    1. Probe Selection
                                                                                                                                                                                                                                                                                                                                                                                                    2. PCR-Based Screening
                                                                                                                                                                                                                                                                                                                                                                                                      1. Primer Design
                                                                                                                                                                                                                                                                                                                                                                                                        1. Pool Screening
                                                                                                                                                                                                                                                                                                                                                                                                          1. Nested Screening
                                                                                                                                                                                                                                                                                                                                                                                                          2. Functional Screening
                                                                                                                                                                                                                                                                                                                                                                                                            1. Complementation Analysis
                                                                                                                                                                                                                                                                                                                                                                                                              1. Expression Screening
                                                                                                                                                                                                                                                                                                                                                                                                                1. Activity Assays
                                                                                                                                                                                                                                                                                                                                                                                                            2. Gene Editing Technologies
                                                                                                                                                                                                                                                                                                                                                                                                              1. CRISPR-Cas9 System
                                                                                                                                                                                                                                                                                                                                                                                                                1. Mechanism of Action
                                                                                                                                                                                                                                                                                                                                                                                                                  1. Cas9 Endonuclease
                                                                                                                                                                                                                                                                                                                                                                                                                    1. Guide RNA Function
                                                                                                                                                                                                                                                                                                                                                                                                                      1. PAM Sequence Recognition
                                                                                                                                                                                                                                                                                                                                                                                                                        1. Double-Strand Break Formation
                                                                                                                                                                                                                                                                                                                                                                                                                        2. Guide RNA Design
                                                                                                                                                                                                                                                                                                                                                                                                                          1. Target Site Selection
                                                                                                                                                                                                                                                                                                                                                                                                                            1. Off-Target Minimization
                                                                                                                                                                                                                                                                                                                                                                                                                              1. Efficiency Optimization
                                                                                                                                                                                                                                                                                                                                                                                                                              2. Applications in Genome Editing
                                                                                                                                                                                                                                                                                                                                                                                                                                1. Gene Knockout
                                                                                                                                                                                                                                                                                                                                                                                                                                  1. Gene Knock-in
                                                                                                                                                                                                                                                                                                                                                                                                                                    1. Base Editing
                                                                                                                                                                                                                                                                                                                                                                                                                                      1. Epigenome Editing
                                                                                                                                                                                                                                                                                                                                                                                                                                    2. Other Gene Editing Tools
                                                                                                                                                                                                                                                                                                                                                                                                                                      1. Zinc Finger Nucleases
                                                                                                                                                                                                                                                                                                                                                                                                                                        1. Zinc Finger Domains
                                                                                                                                                                                                                                                                                                                                                                                                                                          1. FokI Nuclease Domain
                                                                                                                                                                                                                                                                                                                                                                                                                                            1. Target Specificity
                                                                                                                                                                                                                                                                                                                                                                                                                                            2. TALENs
                                                                                                                                                                                                                                                                                                                                                                                                                                              1. TALE Repeat Structure
                                                                                                                                                                                                                                                                                                                                                                                                                                                1. DNA Binding Specificity
                                                                                                                                                                                                                                                                                                                                                                                                                                                  1. Nuclease Activity
                                                                                                                                                                                                                                                                                                                                                                                                                                                  2. Base Editors
                                                                                                                                                                                                                                                                                                                                                                                                                                                    1. Cytosine Base Editors
                                                                                                                                                                                                                                                                                                                                                                                                                                                      1. Adenine Base Editors
                                                                                                                                                                                                                                                                                                                                                                                                                                                        1. Prime Editors

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                                                                                                                                                                                                                                                                                                                                                                                                                                                  8. Epigenetics and Nucleic Acid Modifications

                                                                                                                                                                                                                                                                                                                                                                                                                                                  Go to top

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                                                                                                                                                                                                                                                                                                                                                                                                                                                  1. Introduction to Nucleic Acids