Gene Cloning Chapter Notes | Biology for BMAT (Section 2) PDF Download

Introduction

• Gene cloning is a standard procedure in biotechnology to produce a gene’s product for various applications.
• It involves transferring a DNA fragment containing the gene of interest to a host cell via a vector to create multiple copies for characterization and application.
• Advances in genetic engineering enable DNA analysis, isolation of specific genes, insertion into autonomously replicating vectors (e.g., plasmids) to form recombinant DNA (rDNA), and introduction into hosts (e.g., bacteria) for amplification.
• This process generates a virtually unlimited number of gene copies, referred to as clones.

Identification of Candidate Gene

• Recombinant DNA technology has been used to develop crops resistant to pests, diseases, herbicides, and pathogens by manipulating and transferring specific genes to express desired traits.
• Identifying the candidate gene is a significant challenge due to the complexity of an organism’s genome.
• Gene selection is based on its biomedical, economic, or evolutionary significance, determined through biochemical and physiological studies.
• Examples of candidate genes include those addressing diseases (e.g., insulin deficiency causing diabetes), metabolic defects (e.g., iron deficiency leading to chlorosis in plants), environmental resistance (e.g., salinity tolerance), infection resistance, or economically important traits (e.g., milk proteins, blood clotting factors).
• Searching for a gene of interest is challenging; for instance, a human haploid genome contains approximately 3.2 billion base pairs, making a 3000–3500 bp gene difficult to locate.
• Methods to identify genes include deducing the DNA sequence from the amino acid sequence of a specific polypeptide chain.
• Another approach involves isolating mRNA from specific tissues, synthesizing single-stranded cDNA using reverse transcriptase, and converting it to double-stranded cDNA for cloning.

Isolation of Nucleic Acids

• Isolating nucleic acids is a fundamental requirement for molecular biology experiments, facing challenges due to their low cellular abundance compared to proteins, carbohydrates, and lipids.
• Nucleic acids, particularly DNA, are susceptible to cleavage under harsh physical stress due to their large size.
• The chemical bonds and groups in nucleic acids make them vulnerable to chemical agents.
• Four key steps in nucleic acid extraction include:
• Rupturing cell membranes or walls to release nucleic acids and other cellular molecules.
• Protecting nucleic acids from degrading enzymes (nucleases) released during cell lysis.
• Separating nucleic acids from other cellular molecules.
• Precipitating and concentrating nucleic acids using ethanol or isopropanol.
• Different organisms require specific lysis strategies due to variations in cell boundaries; animal cells have easily disrupted plasma membranes, while plant and bacterial cells have tougher cell walls.
• Methods for cell disruption include homogenization, grinding, sonication, or enzymatic treatment to release nucleic acids, exposing them to nucleases.
• For bacterial DNA isolation, lysozyme digests the peptidoglycan cell wall, and detergents like sodium dodecyl sulphate (SDS) disrupt the lipid bilayer of cell membranes.
• Plant cells are mechanically ruptured using a blender, with cetyl trimethyl ammonium bromide (CTAB) as a cationic detergent to lyse cell walls and separate DNA from polysaccharides and polyphenols.
• CTAB solubility depends on ionic strength: at low ionic strength, DNA is soluble, while polysaccharides are insoluble; at high ionic strength, the reverse occurs.
• Polyvinyl pyrrolidone (PVP) is added to neutralize phenols in plant cell extractions.
• Soluble DNA in the supernatant is extracted with chloroform-isoamyl alcohol, and precipitated using ethanol or isopropanol.
• Animal cell membranes are disrupted by detergents to release intracellular components.
• Extraction media typically use a mild alkaline pH buffer (0.05 M ionic strength) with EDTA to chelate divalent cations (e.g., Mn²⁺, Mg²⁺), preventing nuclease activity and salt formation with nucleic acid phosphate groups.
• SDS, an anionic detergent, makes proteins anionic, dissociating them from nucleic acids and inhibiting nuclease activity.
• High sodium chloride concentrations reduce ionic interactions between DNA and cations, ensuring complete dissociation from proteins.
• Deproteinization uses chloroform and isoamyl alcohol, forming a lower organic layer (denatured proteins) and an upper aqueous layer (nucleic acids) upon centrifugation.
• Chloroform denatures proteins, while isoamyl alcohol stabilizes the organic-aqueous interface.
• Ethanol reduces the polarity of the aqueous medium, precipitating nucleic acids, which are then isolated by centrifugation.
• RNA is removed using ribonuclease A, which digests RNA into ribonucleotides.
• RNA isolation is challenging due to its single-stranded nature and the abundance of ribonucleases (RNases) in the environment.
• Total RNA is extracted using guanidinium isothiocyanate (GITC)-phenol-chloroform, a chaotropic reagent that disrupts hydrogen bonds, denatures proteins, and inactivates RNases.
• Centrifugation separates the solution into an aqueous phase (RNA), an interface (precipitated proteins), and an organic phase (DNA, lipids, proteins).
• RNA is precipitated from the aqueous phase using isopropanol.
• Plasmid DNA is separated from genomic DNA by boiling bacterial lysate, which denatures chromosomal DNA and proteins, forming a gel that is removed by centrifugation.
• Partially denatured plasmid DNA renatures as a circular double helix, remaining soluble.
• Alternatively, bacterial lysis with SDS and NaOH denatures cell contents, and potassium ions precipitate chromosomal DNA and proteins, leaving plasmid DNA in the supernatant for ethanol precipitation.

Enzymes Used for Recombinant DNA Technology

• Enzymes are critical tools in rDNA technology, enabling DNA cutting and ligation for vector and gene manipulation.
• Nucleases hydrolyze phosphodiester bonds between nucleotide sugar residues, categorized as DNases (DNA-specific) or RNases (RNA-specific).
• Nucleases are divided into exonucleases, which remove mononucleotides from the 3’ or 5’ ends, and endonucleases, which cleave internal phosphodiester bonds.
• Restriction endonucleases (REs) are endonucleases that cleave DNA at specific recognition sequences (4–8 bp), primarily found in bacteria and archaea as a defense against bacteriophages.
• REs are classified into three types based on cofactor requirements and cleavage site:
• Type I: Cleave ~1000 bp from the recognition site, require S-adenosyl methionine (SAM), Mg²⁺, and ATP, and have endonuclease, methylase, and ATPase activities (e.g., EcoAI, CfrAI).
• Type II: Cleave within the recognition site, require Mg²⁺, and have only endonuclease activity, widely used in rDNA technology (e.g., EcoRI, BamHI, HindIII).
• Type III: Cleave 24–26 bp from the recognition site, require SAM, Mg²⁺, and ATP, and have endonuclease and methylase activities (e.g., EcoP1, HinfIII, EcoP15I).
• Type II REs recognize palindromic sequences, producing 5’-phosphate and 3’-hydroxyl ends, with cuts yielding either sticky (cohesive) or blunt ends.
• DNA ligase joins DNA strands by catalyzing phosphodiester bond formation, using NAD (E. coli) or ATP (T4 bacteriophage, eukaryotes) as energy sources.
• T4 DNA ligase joins both cohesive and blunt ends, while E. coli DNA ligase joins cohesive ends.
• DNA polymerases synthesize new DNA strands in the 5’→3’ direction using dNTPs, requiring a primer with a free 3’-OH group.
• E. coli DNA polymerase I has additional 5’→3’ and 3’→5’ exonuclease activities.
• Alkaline phosphatase removes the 5’-phosphate group from DNA strands.
• Polynucleotide kinase adds a phosphate group to the 5’-OH end, counteracting alkaline phosphatase.
• Terminal deoxynucleotidyl transferase adds homopolymer tails to the 3’ end without a template.
• Reverse transcriptase (RNA-dependent DNA polymerase) synthesizes cDNA from an RNA template, used in retroviruses.
• Poly A polymerase adds adenine residues to the 3’-OH end of RNA.

Modes of DNA Transfer

• Transferring foreign DNA into host cells (prokaryotic or eukaryotic) is a key step in rDNA technology.
• Natural bacterial DNA uptake occurs via transformation, transduction, or conjugation.
• Transformation involves direct uptake of exogenous DNA through the cell membrane, integrating into the recipient’s genome, creating transformants.
• Transduction occurs when bacteriophages transfer bacterial DNA during infection, forming transductants.
• In the lysogenic cycle, bacteriophage DNA integrates into the bacterial genome, remaining dormant; upon excision, it may carry bacterial DNA, which is packaged into new phage particles.
• Conjugation transfers DNA between bacteria via direct cell-to-cell contact, mediated by conjugative plasmids (e.g., F plasmid), with the donor transferring DNA to the recipient.
• Artificial methods include chemical-based transfection (e.g., calcium chloride, lipofection) and physical transfection (e.g., electroporation, microinjection, biolistics).

Screening and Selection

• Selecting transformed bacteria with recombinant vectors is critical for successful cloning, distinguishing recombinant from non-recombinant cells.
• The success rate of insert integration into plasmids and transformation is low, necessitating efficient screening methods.
• Selection is based on differences in biological traits, such as antibiotic resistance, protein expression (e.g., β-galactosidase, GFP), or nutritional requirements (e.g., leucine dependence).
• Direct selection identifies transformed cells by trait expression, such as antibiotic resistance, allowing only transformed cells to survive in selective media.
• Insertional inactivation uses vectors with two markers (e.g., two antibiotic resistance genes or one antibiotic resistance gene and lacZ) to detect recombinants.
• In insertional inactivation, the insert disrupts one marker (e.g., ampicillin resistance gene), inactivating its expression.
• For example, a plasmid with ampicillin (ampR) and tetracycline (tetR) resistance genes has the insert cloned into ampR, inactivating it.
• Bacteria are plated on tetracycline media to eliminate non-transformed cells, followed by replica plating on ampicillin media to identify colonies lacking ampicillin resistance (recombinants).
• Blue-white selection uses the lacZ gene, which expresses β-galactosidase to cleave X-gal, producing a blue pigment.
• An insert in lacZ disrupts β-galactosidase expression, resulting in white colonies, while non-recombinant cells form blue colonies.
• Alternative screening uses GFP, where an insert disrupts GFP production, resulting in non-fluorescent bacteria.

Blotting Techniques

• Blotting techniques separate and identify DNA, RNA, or proteins from mixtures by immobilizing them on nitrocellulose, nylon, or PVDF membranes.
• Membranes are hydrophobic, chemically resistant, and have high affinity for nucleic acids and proteins.
• Specific molecules are detected using antibodies (proteins) or nucleic acid probes (DNA/RNA) via hybridization, with detection methods including chromogenic, fluorescence, chemiluminescence, or radioactive tags.
• Southern blotting, developed by Edwin Southern, detects specific DNA sequences.
• DNA is cut with restriction endonucleases, separated by agarose gel electrophoresis, transferred to a membrane via capillary action, and hybridized with a labeled probe for detection by autoradiography or fluorescence.
• Northern blotting, developed by J. Alwine, David Kemp, and George Stark, detects specific RNA molecules.
• Total RNA is extracted, separated by agarose gel electrophoresis, transferred to a membrane, and hybridized with radioactive or fluorescent probes, detected by autoradiography.
• Formamide in the transfer buffer lowers the annealing temperature to prevent RNA degradation.
• Western blotting, named by W. Neal Burnette, detects specific proteins using SDS-PAGE for separation, followed by transfer to a membrane and detection with specific antibodies.
• Eastern blotting detects post-translational protein modifications using specific substrates or antibodies.
• Blotting techniques collectively identify a gene (Southern), its RNA transcripts (Northern), and its protein products (Western).

Polymerase Chain Reaction (PCR)

• PCR, developed by Kary B. Mullis, amplifies small DNA amounts into millions of copies, earning Mullis the 1993 Nobel Prize in Chemistry.
• It mimics cellular DNA replication through thermal cycling in a thermocycler, involving heating and cooling.
• PCR uses heat-stable Taq polymerase from Thermus aquaticus or Pfu polymerase from Pyrococcus furiosus for high-fidelity DNA synthesis at 72–78°C.
• Two synthetic primers (forward and reverse) complementary to the target DNA’s flanking sequences provide a 3’-OH starting point for synthesis.
• PCR involves three steps:
• Denaturation at 94–95°C breaks hydrogen bonds, separating double-stranded DNA into single strands.
• Annealing at 50–55°C allows primers to bind to complementary template sites.
• Extension at 72°C enables Taq polymerase to add dNTPs, synthesizing new DNA strands in the 5’→3’ direction.
• Each cycle doubles the DNA, yielding 2ⁿ copies after n cycles (typically 25–30 cycles).
• Amplified products (amplicons) are analyzed by gel electrophoresis for semi-quantitative PCR, with band intensity indicating DNA amount.
• Reverse transcription PCR (RT-PCR) amplifies DNA from mRNA via reverse transcriptase, producing cDNA.
• Real-time quantitative PCR (qPCR) uses fluorescent dyes (e.g., SYBR Green) to measure double-stranded DNA after each cycle, eliminating the need for gel electrophoresis.
• qPCR thermocyclers have detectors to measure fluorescence, proportional to the PCR product.
• PCR applications include quantifying mRNA for gene expression, amplifying DNA from crime scenes or fossils, and detecting SARS-CoV-2 via real-time RT-PCR.
• SARS-CoV-2 RT-PCR tests target ORF1a/b, nucleocapsid (N), or spike protein (S) genes, using probes with reporter and quencher dyes to generate fluorescent signals during amplification.
• The threshold cycle (Ct) value indicates the cycle at which fluorescence exceeds background, inversely proportional to the initial target gene amount.

DNA Libraries

• DNA libraries store cloned DNA fragments in vectors to identify and isolate specific sequences from large genomes (e.g., human diploid genome: ~3×10⁹ bp).
• Genes are randomly arranged, and most genomic DNA is untranscribed, making direct isolation challenging without known sequences.
• Two main types are genomic and cDNA libraries.
• A genomic library contains small DNA fragments representing an organism’s entire genome.
• Construction involves isolating genomic DNA, digesting it with restriction enzymes, ligating fragments into vectors, transforming bacterial hosts, and growing colonies to form the library.
• Applications include genome sequencing, identifying unexpressed genes, studying species evolution, and comparing healthy and diseased tissue sequences.
• A cDNA library contains clones of cDNA synthesized from mRNA of a specific cell type or tissue, reflecting expressed genes.
• Construction involves isolating mRNA, synthesizing cDNA with reverse transcriptase, removing mRNA with RNase, converting single-stranded cDNA to double-stranded cDNA with DNA polymerase, and cloning into vectors.
• cDNA libraries vary by tissue or physiological state due to tissue-specific gene expression (e.g., insulin in pancreatic beta cells, albumin in liver cells).
• Applications include identifying active genes in specific tissues, isolating specific genes, and screening genomic libraries using cDNA probes.