🧬Molecular Biology Unit 8 – Molecular Techniques and Tools

Key Concepts and Terminology

• Molecular biology studies biological processes at the molecular level, focusing on DNA, RNA, and proteins
• Central dogma of molecular biology describes the flow of genetic information from DNA to RNA to proteins
  • DNA serves as the blueprint for cellular processes and is replicated during cell division
  • RNA acts as an intermediary, carrying genetic information from DNA to ribosomes for protein synthesis
  • Proteins perform various functions within cells, including catalyzing reactions, providing structure, and regulating gene expression
• Genome refers to the complete set of genetic material in an organism, including both coding and non-coding regions
• Gene expression is the process by which genetic information is used to synthesize functional gene products, such as proteins or non-coding RNAs
• Transcription is the synthesis of RNA from a DNA template, catalyzed by RNA polymerase enzymes
• Translation is the process of synthesizing proteins from mRNA templates, occurring at ribosomes
• Recombinant DNA technology involves manipulating and combining DNA molecules from different sources to create novel genetic sequences

DNA and RNA Extraction Methods

• DNA and RNA extraction is the process of isolating nucleic acids from biological samples for further analysis
• Cell lysis is the first step in extraction, which involves disrupting cell membranes to release cellular contents
  • Chemical methods use detergents (SDS) or enzymes (lysozyme) to break down cell walls and membranes
  • Mechanical methods include sonication, bead-beating, or freeze-thaw cycles to physically disrupt cells
• Purification steps remove contaminants (proteins, lipids, and carbohydrates) from the lysate to obtain pure nucleic acids
  • Organic extraction uses phenol-chloroform to separate nucleic acids from proteins and other contaminants
  • Silica-based methods rely on the selective binding of nucleic acids to silica matrices in the presence of chaotropic salts
• Precipitation of nucleic acids is achieved by adding ethanol or isopropanol, which reduces their solubility and facilitates pelleting by centrifugation
• Quantification and quality assessment of extracted DNA and RNA are performed using spectrophotometry (NanoDrop) or fluorometry (Qubit)
• RNA extraction requires additional precautions to prevent degradation by ubiquitous RNase enzymes, such as using RNase-free reagents and equipment

Polymerase Chain Reaction (PCR) Techniques

• PCR is a method for amplifying specific DNA sequences exponentially using a thermal cycling process
• PCR requires a DNA template, two primers (forward and reverse) complementary to the target sequence, DNA polymerase (Taq), dNTPs, and buffer
• The PCR process consists of three main steps: denaturation, annealing, and extension
  • Denaturation at high temperature (94-96°C) separates double-stranded DNA into single strands
  • Annealing at a lower temperature (50-65°C) allows primers to bind to their complementary sequences on the template
  • Extension at 72°C enables DNA polymerase to synthesize new DNA strands complementary to the template
• PCR amplification is exponential, doubling the number of target sequences with each cycle
• Reverse transcription PCR (RT-PCR) is used to amplify RNA by first converting it to complementary DNA (cDNA) using reverse transcriptase
• Quantitative PCR (qPCR) or real-time PCR allows for the quantification of target sequences by measuring fluorescence during amplification
  • TaqMan assays use fluorogenic probes that emit fluorescence upon cleavage during extension
  • SYBR Green assays rely on a dye that fluoresces when bound to double-stranded DNA

Gel Electrophoresis and Blotting

• Gel electrophoresis is a technique used to separate nucleic acids or proteins based on their size and charge
• Agarose gel electrophoresis is commonly used for DNA and RNA, while polyacrylamide gel electrophoresis (PAGE) is used for proteins and smaller nucleic acids
• Nucleic acids are negatively charged due to their phosphate backbone and migrate towards the positive electrode when an electric field is applied
• Separation is influenced by the size of the molecules, with smaller fragments migrating faster through the gel matrix
• Visualization of separated DNA or RNA bands is achieved by staining with ethidium bromide or safer alternatives (SYBR Safe), which intercalate between base pairs and fluoresce under UV light
• Blotting techniques transfer separated molecules from the gel onto a membrane for further analysis
  • Southern blotting is used for DNA and involves transferring DNA fragments onto a nylon or nitrocellulose membrane
  • Northern blotting is used for RNA and involves transferring RNA molecules onto a membrane
  • Western blotting is used for proteins and involves transferring proteins onto a PVDF or nitrocellulose membrane

DNA Sequencing Technologies

• DNA sequencing determines the precise order of nucleotides in a DNA molecule
• Sanger sequencing, also known as the chain-termination method, was the first widely used sequencing technology
  • It relies on the incorporation of fluorescently labeled dideoxynucleotides (ddNTPs) during DNA synthesis, which terminate elongation
  • The resulting fragments are separated by capillary electrophoresis, and the fluorescent signals are used to determine the sequence
• Next-generation sequencing (NGS) technologies have revolutionized genomic research by enabling high-throughput and cost-effective sequencing
  • Illumina sequencing (sequencing by synthesis) uses reversible terminator chemistry and fluorescently labeled nucleotides to sequence millions of fragments in parallel
  • Ion Torrent sequencing (semiconductor sequencing) detects pH changes caused by the release of hydrogen ions during nucleotide incorporation
  • Pacific Biosciences (PacBio) and Oxford Nanopore Technologies offer long-read sequencing methods that can sequence several kilobases in a single read
• Whole-genome sequencing (WGS) aims to determine the complete DNA sequence of an organism's genome
• RNA sequencing (RNA-seq) is used to analyze the transcriptome by sequencing cDNA libraries generated from RNA samples

Cloning and Recombinant DNA Techniques

• Cloning involves creating identical copies of a DNA fragment by inserting it into a vector and propagating it in a host cell
• Restriction enzymes are used to cut DNA at specific recognition sites, generating sticky or blunt ends
  • Sticky ends have single-stranded overhangs that can base-pair with complementary ends
  • Blunt ends have no overhangs and can be joined with any other blunt end
• DNA ligase is used to join the insert DNA to the vector by forming phosphodiester bonds between the ends
• Vectors are DNA molecules that can replicate independently and carry the inserted DNA
  • Plasmids are circular DNA molecules commonly used as cloning vectors in bacteria
  • Viral vectors (retroviruses, adenoviruses) are used for gene delivery and expression in mammalian cells
• Transformation is the process of introducing recombinant DNA into a host cell, such as bacteria (E. coli) or yeast (S. cerevisiae)
• Selection markers (antibiotic resistance genes) are used to identify and isolate cells containing the recombinant DNA
• Blue-white screening is a technique used to identify successful cloning events based on the disruption of the lacZ gene in the vector

Gene Expression Analysis Tools

• Gene expression analysis tools are used to study the levels and patterns of gene expression in cells or tissues
• Northern blotting is used to detect and quantify specific RNA molecules by hybridization with labeled probes
• Reverse transcription PCR (RT-PCR) and quantitative PCR (qPCR) are used to measure gene expression by amplifying cDNA derived from RNA
• DNA microarrays are used to simultaneously analyze the expression of thousands of genes
  • cDNA or oligonucleotide probes specific to different genes are immobilized on a solid surface
  • Fluorescently labeled cDNA from sample and reference RNA is hybridized to the array, and the relative fluorescence intensities indicate gene expression levels
• RNA sequencing (RNA-seq) provides a comprehensive and quantitative analysis of the transcriptome
  • It involves converting RNA to cDNA, followed by high-throughput sequencing and mapping of the reads to a reference genome or transcriptome
  • RNA-seq can identify novel transcripts, alternative splicing events, and gene fusion events
• In situ hybridization (ISH) is used to detect and localize specific RNA or DNA sequences within intact cells or tissues using labeled probes
• Reporter gene assays are used to study promoter activity and gene regulation by linking a reporter gene (GFP, luciferase) to a promoter of interest

Applications in Research and Medicine

• Molecular techniques have revolutionized our understanding of biological processes and have numerous applications in research and medicine
• Genetic engineering involves modifying the genetic material of an organism to alter its characteristics or produce desired substances
  • Recombinant proteins (insulin, growth hormones) are produced by inserting the corresponding genes into bacterial or mammalian cells
  • Genetically modified organisms (GMOs) are created by introducing foreign genes to confer desirable traits (herbicide resistance in crops, disease resistance in animals)
• Gene therapy aims to treat genetic disorders by introducing functional copies of defective genes into patient cells
  • Ex vivo gene therapy involves modifying cells outside the body and then transplanting them back into the patient
  • In vivo gene therapy involves delivering the therapeutic gene directly into the patient's cells using viral or non-viral vectors
• Molecular diagnostics use molecular techniques to detect and identify pathogens, genetic disorders, or cancer biomarkers
  • PCR-based tests are used to detect infectious agents (HIV, SARS-CoV-2) or genetic mutations (sickle cell anemia, cystic fibrosis)
  • DNA sequencing is used to identify genetic variations associated with diseases or drug responses (pharmacogenomics)
• Personalized medicine aims to tailor medical treatments to an individual's genetic profile to optimize efficacy and minimize adverse effects
• CRISPR-Cas9 is a revolutionary gene-editing tool that allows precise modification of DNA sequences in living cells
  • It consists of a guide RNA that directs the Cas9 endonuclease to a specific DNA sequence, where it creates a double-strand break
  • The break can be repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR), allowing for gene knockouts or precise edits, respectively