BIO 408 — Applied Biotechnology: Lecture Note

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BIO la-NET | ONLINE LEARNING VIDEO PLATFORM FOR POST-SECONDARY & HIGHER INSTITUTION STUDENTS

BIO la-NET | ONLINE LEARNING VIDEO PLATFORM FOR POST-SECONDARY & HIGHER INSTITUTION STUDENTS Course code: BIO 408 Course title: Applied Biotechnology Course credit load: 2 Units Series 1: DNA and Protein Extraction • Part 1.1: Principles of Cell Disruption and Tissue Preparation o Sub Part 1.1.1: Overview of extraction: aims, challenges, and tissue types o Sub Part 1.1.2: Mechanical and chemical cell disruption methods o Sub Part 1.1.3: Buffers, reagents, and safety considerations • Part 1.2: DNA Extraction o Sub Part 1.2.1: Principles of DNA extraction o Sub Part 1.2.2: CTAB method for plant DNA extraction o Sub Part 1.2.3: Extraction from animal tissues, blood, and microorganisms • Part 1.3: Protein Extraction o Sub Part 1.3.1: Principles of protein extraction and solubilisation o Sub Part 1.3.2: Extraction from plant and animal tissues o Sub Part 1.3.3: Subcellular fractionation • Part 1.4: Quality Assessment of Extracted Nucleic Acids and Proteins o Sub Part 1.4.1: Visual and spectrophotometric assessment of DNA quality o Sub Part 1.4.2: Assessment of protein yield and integrity o Sub Part 1.4.3: Storage and handling of extracted biomolecules

BIO la-NET | ONLINE LEARNING VIDEO PLATFORM FOR POST-SECONDARY & HIGHER INSTITUTION STUDENTS Introduction Extraction of nucleic acids and proteins from biological tissues is the starting point for virtually all molecular biotechnology applications, from PCR amplification and DNA sequencing to enzyme characterisation and recombinant protein production. The quality and yield of the extracted molecules directly determine the success of downstream analytical and preparative procedures. A good extraction must release sufficient quantities of the target molecule, remove contaminants (proteins, polysaccharides, pigments, lipids, and inhibitors), and preserve the structural integrity of the molecule. This Series introduces the principles and practice of DNA and protein extraction from diverse biological tissues encountered in Nigerian research contexts: crop plants (cassava, maize, cowpea), livestock tissues (muscle, blood, liver), and microorganisms (bacteria, fungi). It covers cell disruption methods, extraction reagents and buffers, standard protocols (including the CTAB method for plant DNA), subcellular fractionation for protein extraction, and quality assessment of the extracted biomolecules. Series Learning Outcomes Upon completing this Series, you should be able to: • SLO 1.1 describe the aims of biomolecule extraction and compare mechanical and chemical cell disruption methods (Linked to Part 1.1), • SLO 1.2 explain the roles of buffers, detergents, and other key reagents used in DNA and protein extraction (Linked to Part 1.1), • SLO 1.3 describe the principles of DNA extraction and outline the steps of the CTAB method for plant tissues (Linked to Part 1.2), • SLO 1.4 describe methods for extracting DNA from animal tissues, blood, and microorganisms (Linked to Part 1.2), • SLO 1.5 explain the principles of protein extraction and subcellular fractionation (Linked to Part 1.3), and

BIO la-NET | ONLINE LEARNING VIDEO PLATFORM FOR POST-SECONDARY & HIGHER INSTITUTION STUDENTS • SLO 1.6 assess the quality and integrity of extracted DNA and protein using spectrophotometric and gel-based methods (Linked to Part 1.4). 1.1 Principles of Cell Disruption and Tissue Preparation 1.1.1 Overview of extraction: aims, challenges, and tissue types The aim of biomolecule extraction is to release the target molecule from the cellular environment in sufficient quantity and purity for the intended downstream application. The principal challenges vary with tissue type: plant tissues present a tough cell wall (cellulose and pectin), large vacuoles containing phenolic compounds and polysaccharides that co-precipitate with DNA and inhibit downstream enzymes, and plastids containing chloroplast DNA that may contaminate nuclear DNA preparations. Animal tissues are generally easier to disrupt but contain proteases and nucleases that rapidly degrade the target molecules if not inhibited promptly. Microbial cells may have rigid peptidoglycan cell walls (bacteria) or thick glucan-chitin walls (fungi) requiring more aggressive disruption. Tissue preparation before extraction includes: freshness (tissues processed immediately or snap-frozen in liquid nitrogen to prevent nuclease and protease degradation); homogenisation (reducing the tissue to a fine powder using liquid nitrogen and a mortar and pestle, or a bead mill for small volumes); removal of non-target material (de-fatting, removal of connective tissue, washing of blood cells); and young, actively growing tissue selection (for plant DNA extraction, young leaves have lower polysaccharide and phenolic content than mature or senescing leaves). For Nigerian crops, the optimal tissue for DNA extraction is young, fully expanded leaf material collected in the morning before significant photosynthetic activity has loaded leaves with starch. Fill in the blank: For plant DNA extraction, young ________ tissue is preferred because it has lower polysaccharide, phenolic, and starch content than mature or senescing tissue, reducing co-precipitation of inhibitors with the extracted DNA.

BIO la-NET | ONLINE LEARNING VIDEO PLATFORM FOR POST-SECONDARY & HIGHER INSTITUTION STUDENTS 1.1.2 Mechanical and chemical cell disruption methods Mechanical disruption physically breaks open cells by applying force. Methods include: mortar and pestle with liquid nitrogen (freezing the tissue in liquid nitrogen makes it brittle; grinding produces a fine powder; standard for plant and tough animal tissues); bead milling (tissue and small glass or ceramic beads are agitated at high speed; suitable for microorganisms, fungi, and small volumes of plant material; instruments include TissueLyser and FastPrep); sonication (high-frequency ultrasound generates cavitation bubbles that lyse cells; used for bacterial and mammalian cell suspensions; generates heat that must be controlled by cooling); and French press (suspension forced through a narrow valve at high pressure; used for bacteria and yeast). Chemical disruption uses reagents to solubilise or weaken cell walls and membranes. Detergents (sodium dodecyl sulphate, SDS; cetyltrimethylammonium bromide, CTAB; Triton X-100; NP-40) solubilise lipid bilayers and denature proteins, releasing cell contents. Chaotropic agents (guanidinium thiocyanate; guanidinium hydrochloride) disrupt hydrogen bonds and hydrophobic interactions, denaturing proteins and releasing nucleic acids; widely used in commercial DNA extraction kits and spin-column protocols. Alkaline lysis (NaOH + SDS) is used for plasmid DNA extraction from bacteria: SDS disrupts the membrane; NaOH denatures chromosomal DNA and proteins; neutralisation with potassium acetate selectively precipitates denatured chromosomal DNA and proteins while the smaller covalently closed circular plasmid DNA remains in solution. Fill in the blank: In alkaline lysis for plasmid extraction, ________ (NaOH + SDS) disrupts bacterial membranes and denatures chromosomal DNA; neutralisation with potassium acetate selectively precipitates chromosomal DNA and proteins, leaving plasmid DNA in the supernatant. 1.1.3 Buffers, reagents, and safety considerations Extraction buffers serve three main functions: maintaining pH to prevent acid or alkaline degradation of the target molecule; providing ionic conditions that stabilise macromolecular structure; and containing chelating agents (EDTA; ethylene diamine tetraacetic acid) that sequester divalent cations (Mg2+, Ca2+) required by nucleases and proteases, inhibiting their

BIO la-NET | ONLINE LEARNING VIDEO PLATFORM FOR POST-SECONDARY & HIGHER INSTITUTION STUDENTS activity. A typical DNA extraction buffer for plant tissue contains: Tris-HCl (pH 8.0; buffering); NaCl (0.1 to 0.8 M; ionic stabilisation); EDTA (20 to 25 mM; DNase inhibition); CTAB or SDS (detergent; cell membrane disruption); and beta-mercaptoethanol or dithiothreitol (DTT; reducing agents that prevent oxidation of phenolic compounds to quinones, which otherwise bind irreversibly to DNA). Beta-mercaptoethanol is highly toxic and volatile; handling must be in a fume hood. Protease inhibitors added to protein extraction buffers include: phenylmethylsulfonyl fluoride (PMSF; serine protease inhibitor; unstable in aqueous solution; added just before use); EDTA (metalloprotease inhibitor); pepstatin A (aspartic protease inhibitor); leupeptin (cysteine and serine protease inhibitor); and complete protease inhibitor cocktail tablets (Roche; convenient combination product). Safety considerations for common extraction reagents: phenol-chloroform (used in classical DNA extraction; highly toxic; causes chemical burns; volatile; must be handled in fume hood with nitrile gloves and eye protection; waste disposed as halogenated organic solvent); chloroform (toxic; anaesthetic; handle in fume hood); liquid nitrogen (extreme cold; risk of asphyxiation in enclosed spaces; use with cryogenic gloves and face shield); SDS (irritant; produces dust; avoid inhalation). Fill in the blank: ________ (EDTA) is added to DNA extraction buffers to chelate divalent cations such as Mg2+ and Ca2+; this inhibits nucleases, which require these metal ions as cofactors, thereby protecting extracted DNA from degradation during the extraction procedure. Pilot questions 1.1 1. State three challenges specific to DNA extraction from plant tissues and explain how each is addressed. 2. Distinguish between mechanical and chemical cell disruption and give two examples of each. 3. State the function of each component in a typical plant DNA extraction buffer: Tris-HCl, NaCl, EDTA, CTAB, and beta-mercaptoethanol. 4. Explain the principle of alkaline lysis and why it selectively recovers plasmid DNA rather than chromosomal DNA.

BIO la-NET | ONLINE LEARNING VIDEO PLATFORM FOR POST-SECONDARY & HIGHER INSTITUTION STUDENTS 5. State four safety precautions that must be observed when working with phenol-chloroform extraction reagents. 1.2 DNA Extraction 1.2.1 Principles of DNA extraction DNA extraction aims to release genomic or plasmid DNA from cells, separate it from proteins, RNA, polysaccharides, and other contaminants, and recover it in a form pure and concentrated enough for downstream applications (PCR, restriction digestion, sequencing, southern blotting). The fundamental steps common to all DNA extraction methods are: (1) cell disruption (described in Part 1.1); (2) protein denaturation and removal (by heat, detergent, proteinase K digestion, or

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