🧬Bioinformatics Unit 1 – Fundamentals of molecular biology

Key Concepts and Terminology

• Central dogma of molecular biology describes the flow of genetic information from DNA to RNA to proteins
• Nucleotides serve as the building blocks of DNA and RNA consisting of a sugar, phosphate group, and nitrogenous base
• DNA double helix structure discovered by Watson and Crick in 1953 using X-ray crystallography data from Rosalind Franklin
  • Consists of two antiparallel strands held together by hydrogen bonds between complementary base pairs (A-T and G-C)
• Genes are segments of DNA that encode specific proteins or functional RNA molecules
• Genome refers to the complete set of genetic material in an organism
• Transcription process of synthesizing RNA from a DNA template catalyzed by RNA polymerase
• Translation process of synthesizing proteins from an mRNA template by ribosomes
• Genetic code determines the relationship between codons (triplets of nucleotides) and amino acids in protein synthesis

DNA Structure and Function

• DNA (deoxyribonucleic acid) stores and transmits genetic information in living organisms
• Composed of four nucleotide bases: adenine (A), thymine (T), guanine (G), and cytosine (C)
  • A pairs with T and G pairs with C through hydrogen bonding
• Sugar-phosphate backbone provides structural stability and connects nucleotides
• Major and minor grooves in the double helix allow for protein interactions and recognition
• Supercoiling of DNA facilitates compact packaging in chromosomes
  • Histones are proteins that help organize and condense DNA into chromatin
• DNA replication is the process of copying genetic material before cell division
  • Semiconservative replication each new double helix contains one original strand and one newly synthesized strand
  • DNA polymerases catalyze the addition of nucleotides to the growing strand
• DNA repair mechanisms (mismatch repair, base excision repair) maintain genetic integrity by correcting errors and damage

RNA and Protein Synthesis

• RNA (ribonucleic acid) is a single-stranded molecule that plays various roles in gene expression
• Three main types of RNA: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA)
  • mRNA carries genetic information from DNA to ribosomes for protein synthesis
  • tRNA transfers specific amino acids to the growing polypeptide chain during translation
  • rRNA is a component of ribosomes and catalyzes peptide bond formation
• Transcription is the synthesis of RNA from a DNA template by RNA polymerase
  • Initiated at promoter regions and terminated at specific sequences
  • Eukaryotic mRNA undergoes post-transcriptional modifications (5' capping, 3' polyadenylation, splicing)
• Translation is the synthesis of proteins from an mRNA template by ribosomes
  • Occurs in three stages: initiation, elongation, and termination
  • Genetic code determines the relationship between codons and amino acids
    • 64 possible codons, 61 coding for amino acids and 3 stop codons
• Post-translational modifications (phosphorylation, glycosylation) can alter protein function and stability

Gene Regulation and Expression

• Gene expression is the process by which genetic information is used to synthesize functional gene products (proteins or RNA)
• Prokaryotic gene regulation often involves operons, which are clusters of genes under the control of a single promoter
  • Lac operon in E. coli is a classic example of negative regulation by the lac repressor
• Eukaryotic gene regulation is more complex and occurs at multiple levels
  • Chromatin structure and histone modifications (acetylation, methylation) affect gene accessibility
  • Transcription factors bind to specific DNA sequences (enhancers, silencers) to regulate transcription
  • Alternative splicing of pre-mRNA can generate multiple protein isoforms from a single gene
  • RNA interference (RNAi) can silence gene expression through the action of small non-coding RNAs (siRNA, miRNA)
• Epigenetic modifications are heritable changes in gene expression without altering the DNA sequence
  • DNA methylation and histone modifications are examples of epigenetic mechanisms
• Gene regulatory networks involve the coordinated expression of multiple genes in response to environmental or developmental cues

Molecular Biology Techniques

• Polymerase chain reaction (PCR) amplifies specific DNA sequences using primers, dNTPs, and DNA polymerase
  • Real-time PCR (qPCR) quantifies the amplification of target sequences in real-time using fluorescent probes
• DNA sequencing determines the precise order of nucleotides in a DNA molecule
  • Sanger sequencing is a traditional method based on dideoxy chain termination
  • Next-generation sequencing (NGS) technologies enable high-throughput, parallel sequencing of millions of DNA fragments
• Cloning involves the insertion of a DNA fragment into a vector (plasmid, viral vector) for propagation in a host cell
  • Restriction enzymes and DNA ligase are used for cutting and joining DNA fragments
• Gel electrophoresis separates DNA, RNA, or proteins based on size and charge in an agarose or polyacrylamide gel matrix
• Southern blotting detects specific DNA sequences using labeled probes after transfer to a membrane
• Northern blotting detects specific RNA sequences using labeled probes after transfer to a membrane
• Western blotting detects specific proteins using antibodies after transfer to a membrane

Bioinformatics Tools for Molecular Biology

• Sequence alignment tools (BLAST, CLUSTAL) compare and analyze DNA or protein sequences to identify similarities and evolutionary relationships
• Genome browsers (UCSC Genome Browser, Ensembl) provide interactive visualization and annotation of genomic data
• Gene expression databases (GEO, ArrayExpress) store and analyze microarray and RNA-seq data to study gene expression patterns
• Protein structure databases (PDB, UniProt) contain information on the 3D structure and function of proteins
• Pathway analysis tools (KEGG, Reactome) integrate and visualize molecular interactions and biological processes
• Variant annotation tools (ANNOVAR, VEP) predict the functional impact of genetic variants on protein function
• Machine learning algorithms (support vector machines, neural networks) can be applied to various molecular biology problems (e.g., predicting protein-protein interactions, identifying regulatory elements)

Applications in Research and Medicine

• Personalized medicine tailors medical treatments to an individual's genetic profile
  • Pharmacogenomics studies how genetic variations affect drug response and toxicity
• Genetic testing can identify inherited disorders, predict disease risk, and guide treatment decisions
  • BRCA1/2 testing for hereditary breast and ovarian cancer risk is a well-known example
• Gene therapy aims to treat or prevent diseases by introducing functional genes into cells
  • Approved treatments for rare disorders (Leber congenital amaurosis, spinal muscular atrophy)
• Genome editing technologies (CRISPR-Cas9, TALENs) enable precise modification of DNA sequences
  • Potential applications in correcting genetic defects, creating disease models, and agricultural biotechnology
• Synthetic biology involves the design and construction of novel biological systems or organisms
  • Applications in biofuel production, biosensor development, and drug manufacturing
• Molecular diagnostics use molecular biology techniques to detect and monitor diseases
  • PCR-based tests for infectious diseases (COVID-19, HIV), cancer biomarkers, and genetic disorders

Challenges and Future Directions

• Ethical considerations surrounding genetic testing, gene therapy, and genome editing
  • Informed consent, privacy, and potential for misuse or discrimination
• Technical limitations in sequencing and analyzing complex genomic regions (repetitive sequences, structural variations)
• Data storage and management challenges associated with the increasing volume of genomic data
  • Need for efficient algorithms, data compression, and secure storage solutions
• Integration of multi-omics data (genomics, transcriptomics, proteomics, metabolomics) to gain a comprehensive understanding of biological systems
• Translating basic research findings into clinical applications and personalized treatments
  • Overcoming barriers in drug development, regulatory approval, and healthcare implementation
• Addressing health disparities and ensuring equitable access to molecular biology-based technologies and treatments
• Fostering interdisciplinary collaborations between molecular biologists, bioinformaticians, clinicians, and other stakeholders to advance the field