Genomics & Proteomics Basics

Cheatsheet Content

### Unit I: Genome and Manipulation of Genetic Material #### Organisation of Genomes - **Prokaryotic Genome:** - Circular chromosome, typically single copy. - Located in nucleoid region, no membrane-bound nucleus. - Often contains plasmids (extrachromosomal DNA). - Relatively small size, high gene density. - **Eukaryotic Genome:** - Linear chromosomes, multiple copies (diploid). - Located in membrane-bound nucleus. - Contains introns (non-coding) and exons (coding). - Larger size, lower gene density compared to prokaryotes. - Organellar genomes (mitochondrial, chloroplast) also present. - **Plasmids:** - Small, circular, double-stranded DNA molecules distinct from the chromosomal DNA. - Replicate independently. - Carry genes for antibiotic resistance, virulence factors, etc. - Used extensively as vectors in genetic engineering. #### DNA Cloning Basics - **Definition:** Process of making multiple, identical copies of a specific DNA sequence. - **Key Steps:** 1. **Isolation:** Obtain target DNA and vector (e.g., plasmid). 2. **Restriction Digestion:** Cut target DNA and vector with same restriction enzyme. 3. **Ligation:** Join target DNA into vector using DNA ligase. 4. **Transformation:** Introduce recombinant plasmid into host cell (e.g., bacteria). 5. **Selection/Screening:** Identify host cells containing the recombinant plasmid. #### Polymerase Chain Reaction (PCR) - **Definition:** _In vitro_ method to amplify specific DNA sequences exponentially. - **Components:** DNA template, primers, Taq polymerase, dNTPs, buffer. - **Cycles (3 steps):** 1. **Denaturation:** Heat to separate DNA strands (~$94-98^\circ C$). 2. **Annealing:** Cool to allow primers to bind to complementary sequences (~$50-65^\circ C$). 3. **Extension:** Heat to optimal temperature for Taq polymerase to synthesize new DNA strands (~$72^\circ C$). - **Applications:** Gene cloning, DNA fingerprinting, disease diagnosis. #### DNA Fingerprinting - **Definition:** Technique to identify individuals based on unique patterns in their DNA. - **Methods:** - **RFLP (Restriction Fragment Length Polymorphism):** Uses restriction enzymes to cut DNA, then separates fragments by gel electrophoresis. - **STR (Short Tandem Repeats):** Analyzes variations in short, repeated DNA sequences. Highly polymorphic and commonly used. - **Applications:** Forensic science, paternity testing, identification of genetic diseases. #### DNA Sequencing - **Sanger Sequencing (Chain-Termination Method):** - Uses dideoxynucleotides (ddNTPs) which lack a 3'-OH group, terminating DNA synthesis. - Generates fragments of varying lengths, separated by size (e.g., capillary electrophoresis). - Each ddNTP is fluorescently labeled with a different color. - Readout: Sequence determined by the order of fluorescent signals. - **Limitations:** Low throughput, relatively slow. - **Pyrosequencing:** - Detects pyrophosphate release upon nucleotide incorporation. - Uses enzymes (ATP sulfurylase, luciferase) to convert pyrophosphate into light signal. - Nucleotides are added one at a time. - **Advantages:** Real-time detection, good for short reads. #### Tools for Genome Analysis - **Restriction Mapping:** - Uses restriction enzymes to cut DNA at specific recognition sites. - Fragments are separated by gel electrophoresis. - Determines the relative positions of restriction sites on a DNA molecule. - Used to construct physical maps of genomes or plasmids. - **RFLP (Restriction Fragment Length Polymorphism):** - Variations in DNA sequences that create or abolish restriction enzyme recognition sites. - Leads to differences in fragment lengths after restriction digestion. - Detected by Southern blotting. - Used for genetic mapping, disease gene linkage, DNA fingerprinting. ### Unit II: Genome Analysis #### Physical and Genetic Maps - **Genetic Map (Linkage Map):** - Based on recombination frequencies between genes. - Units: centimorgans (cM). - Shows relative order and distance of genes on a chromosome. - Does not represent actual physical distances. - **Physical Map:** - Based on actual physical distances between markers (e.g., base pairs). - Units: base pairs (bp), kilobase pairs (kb), megabase pairs (Mb). - Constructed using techniques like restriction mapping, FISH, sequencing. - Provides a more accurate representation of genome organization. #### Definition of Genomics - **Genomics:** The study of the entire genome of an organism, including its structure, function, evolution, and mapping. - **Goals:** Understand the complete set of genes, their interactions, and their influence on an organism's traits. #### Comparative Genomics - **Definition:** Comparison of genome sequences from different species. - **Purpose:** Identify conserved regions, evolutionary relationships, gene function, and disease mechanisms. - **Insights:** Reveals shared ancestry, unique adaptations, and gene duplication events. #### Functional Genomics - **Definition:** Study of the functions of genes and their products (proteins). - **Techniques:** Transcriptomics (RNA-seq), proteomics (mass spectrometry), metabolomics. - **Goal:** Understand how genes contribute to biological processes and phenotypes. #### Genome Sequencing - **Human Genome Project (HGP):** - International research effort to determine the sequence of the human genome. - Completed in 2003. - Provided the first comprehensive map of the human genetic blueprint. - **Impact:** Revolutionized biology and medicine, enabled personalized medicine. - **Shotgun Sequencing:** - Randomly break genomic DNA into small fragments. - Sequence each fragment. - Assemble overlapping fragments using computational algorithms to reconstruct the entire genome. - **Advantage:** Faster, more efficient for smaller genomes. - **Hierarchical (Clone-Contig) Sequencing:** - Create a physical map of the genome first using large DNA clones (e.g., BACs). - Arrange clones in order, then sequence each clone independently using shotgun approach. - **Advantage:** Better for large, complex genomes with repetitive regions, less computational burden during assembly. #### Next-Generation Sequencing (NGS) Techniques - **General Principle:** Massively parallel sequencing, enabling rapid and cost-effective sequencing of entire genomes or specific regions. - **Illumina Sequencing (Sequencing by Synthesis):** - DNA fragments are attached to a flow cell, amplified to form clonal clusters. - Fluorescently labeled nucleotides are added one at a time, and emission is captured. - Reversible terminators ensure only one nucleotide is added at a time. - **Characteristics:** High accuracy, high throughput, short to medium read lengths. - **Oxford Nanopore Sequencing:** - DNA strands pass through a protein nanopore embedded in a membrane. - Changes in electrical current across the pore are detected as different bases pass through. - **Characteristics:** Long read lengths (up to Mb), real-time sequencing, portable devices. - **Limitations:** Higher error rate than Illumina (though improving), requires more specialized bioinformatics. ### Unit III: Protein Measurement and Separation #### Physical Interactions that Determine the Property of Proteins - **Primary Structure:** Amino acid sequence. Determines all higher-order structures. - **Secondary Structure:** Local folding patterns (e.g., $\alpha$-helices, $\beta$-sheets) stabilized by hydrogen bonds between backbone atoms. - **Tertiary Structure:** Overall 3D shape of a single polypeptide chain, stabilized by various interactions (hydrophobic interactions, ionic bonds, hydrogen bonds, disulfide bridges). - **Quaternary Structure:** Arrangement of multiple polypeptide chains (subunits) in a multi-subunit protein, stabilized by similar interactions as tertiary structure. - **Other Interactions:** - **Hydrophobic Effect:** Nonpolar side chains cluster together away from water. - **Electrostatic Interactions:** Attraction/repulsion between charged amino acid side chains (salt bridges). - **Van der Waals Forces:** Weak, transient interactions between atoms. - **Hydrogen Bonding:** Between polar side chains or with water. - **Disulfide Bonds:** Covalent bonds between cysteine residues. - These interactions influence protein solubility, stability, folding, and function. #### Analytical Techniques to Study Proteins - **Sedimentation Analysis (Ultracentrifugation):** - **Principle:** Proteins are subjected to a strong centrifugal force, causing them to sediment at a rate dependent on their mass, shape, and density. - **Equipment:** Ultracentrifuge. - **Applications:** Determine molecular weight, shape, purity, and analyze protein-protein interactions. - **Svedberg Unit (S):** Unit for sedimentation coefficient, reflects sedimentation rate. - **Gel Filtration Chromatography (Size Exclusion Chromatography):** - **Principle:** Separates proteins based on their size/hydrodynamic volume. - **Matrix:** Porous beads with defined pore sizes. - **Mechanism:** - **Large proteins:** Cannot enter the pores, elute quickly (shortest path). - **Small proteins:** Enter pores, spend more time inside, elute later (longer path). - **Applications:** Determine molecular weight, separate protein mixtures, desalt samples, buffer exchange.