Biotechnology: Class 12 — Chapter 3: Gene Cloning

DNA extraction requires cell lysis, protection from nucleases, separation from other macromolecules, and precipitation.

1. Cell Lysis:
   • Bacteria: Treated with lysozyme to digest peptidoglycan cell walls, followed by SDS (sodium dodecyl sulphate) to disrupt lipid bilayers.
   • Plants: Mechanically ruptured in a blender; treated with CTAB (cetyl trimethyl ammonium bromide), a cationic detergent. CTAB solubility depends on ionic strength: DNA is soluble in CTAB at low ionic strength (polysaccharides insoluble), while polysaccharides are soluble at high ionic strength (DNA insoluble). PVP (polyvinyl pyrrolidone) is added to neutralize polyphenols.
   • Animals: Lysis buffer containing SDS easily disrupts the plasma membrane.
2. Nuclease Inhibition: Lysis buffers maintain a mild alkaline pH and contain EDTA (ethylene diamine tetraacetic acid) to chelate divalent cations like $\text{Mn}^{2+}$ and $\text{Mg}^{2+}$, preventing nuclease activity.
3. Deproteinisation: Exposing the lysate to non-polar solvents like chloroform and isoamyl alcohol. Centrifugation yields three layers: a lower organic phase (denatured proteins), an interface (lysed proteins), and an upper aqueous phase (nucleic acids).
4. Precipitation: Adding chilled ethanol or isopropanol to reduce polarity, rendering nucleic acids insoluble. RNA is removed by adding ribonuclease A (RNase A).

RNA is single-stranded and highly vulnerable to degradation by abundant environmental RNases.

• GITC Reagent: Total RNA is extracted using guanidinium isothiocyanate (GITC)-phenol-chloroform. GITC is an acidic chaotropic agent that disrupts hydrogen bonds, denaturing proteins and inactivating RNases.
• Phase Separation: Acidic pH retains DNA and denatured proteins in the organic phase/interface, while total RNA partitions into the upper aqueous phase.
• Precipitation: RNA is recovered from the aqueous phase using isopropanol.

Nucleases cleave nucleic acids by hydrolysing phosphodiester bonds.

• Exonucleases: Remove nucleotides sequentially from the $3'$ or $5'$ ends of a polynucleotide chain.
• Endonucleases: Cleave internal phosphodiester bonds within DNA or RNA.
• Restriction Endonucleases (REs): Bacterial defense enzymes that recognize specific palindromic sequences ($4 \text{ to } 8$ bp) and cleave DNA.

Restriction enzymes are named systematically based on their origin of isolation:

• First Letter: Genus of the donor microorganism (capitalized, e.g., E for Escherichia).
• Second & Third Letters: Species of the microorganism (lowercase, e.g., co for coli).
• Fourth Letter: Specific strain designation (e.g., R for strain Ry13).
• Roman Numeral: Order of identification/isolation from that strain (e.g., I).

• DNA Ligase: Catalyzes phosphodiester bond formation between adjacent $5'\text{-phosphate}$ and $3'\text{-hydroxyl}$ groups. Bacterial ligase (E. coli) utilizes $\text{NAD}^+$ hydrolysis, whereas bacteriophage ligase (T4 DNA Ligase) utilizes $\text{ATP}$ and can join both cohesive and blunt ends.
• DNA Polymerases: Synthesize complementary DNA strands in the $5' \rightarrow 3'$ direction. DNA Polymerase I of E. coli also exhibits $5' \rightarrow 3'$ and $3' \rightarrow 5'$ exonuclease activities.
• Alkaline Phosphatase: Removes terminal $5'\text{-phosphate}$ groups from linearized vector DNA to prevent self-ligation.
• Polynucleotide Kinase: Transfers a phosphate group from ATP to a free $5'\text{-hydroxyl}$ terminus of DNA.
• Terminal Transferase: Adds homopolymer tails (e.g., poly-A or poly-T) to the $3'$ ends of DNA templates without requiring a complementary template.
• Reverse Transcriptase: An RNA-directed DNA polymerase that synthesizes complementary DNA (cDNA) from an RNA template.
• Poly A Polymerase: Incorporates adenine residues to the $3'\text{-hydroxyl}$ terminus of RNA molecules.

1. Transformation: Direct uptake and integration of exogenous, naked DNA from the surrounding environment across the cell membrane. Recipient cells are called transformants.
2. Transduction: Bacteriophage-mediated transfer of bacterial DNA. In the lysogenic cycle, the phage genome integrates into the host chromosome; upon excision, it may package adjacent bacterial genes and transfer them to a new host (transductants).
3. Conjugation: Direct physical transfer of genetic material (F plasmid) between bacterial cells via a specialized protein tube called a conjugation pilus.

• Direct Selection: Transformed cells are identified by growing them on media containing an antibiotic. Only cells harboring plasmids with the corresponding resistance gene survive.
• Insertional Inactivation: Cloning a gene of interest directly into a selection marker gene, disrupting its sequence and inactivating its function.

Limitation: Distinguishing between recombinant and non-recombinant plasmids using antibiotic replica plating.

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Engineering Methodology & Mechanism: A plasmid vector containing two antibiotic resistance markers ($amp^R$ and $tet^R$) is used. The target gene is cloned into the restriction site within the $amp^R$ gene, inactivating it.
1. Transformants are plated on media containing tetracycline; non-transformants are eliminated.
2. Surviving colonies are replica-plated onto media containing ampicillin using a sterile nitrocellulose membrane.
3. Recombinant colonies (which are ampicillin-sensitive due to insertional inactivation of $amp^R$) fail to grow on the ampicillin plate. Comparing the master plate (tetracycline) and replica plate (ampicillin) identifies the recombinant colonies.

A rapid, single-plate screening method utilizing the lacZ reporter gene.

• Mechanism: The vector contains a lacZ gene encoding the enzyme $\beta$-galactosidase. $\beta$-galactosidase cleaves the colorless chromogenic substrate X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactoside) to form an insoluble blue pigment (5,5′-dibromo-4,4′-dichloro-indigo).
• Recombinant Identification: Insertion of the target DNA into the cloning site within lacZ disrupts the gene. Recombinant colonies cannot produce active $\beta$-galactosidase and remain white, while non-recombinant colonies turn blue.
• Alternative Reporter: Green Fluorescent Protein (GFP); insertional inactivation of the GFP gene results in non-fluorescing recombinant colonies.

Developed by Kary B. Mullis, PCR exponentially amplifies specific DNA sequences through repeated thermal cycles. The number of DNA copies generated after $n$ cycles is $2^n$.

1. Denaturation: Heating the reaction mixture to $94\text{ to }95^\circ\text{C}$ to break hydrogen bonds between complementary strands, yielding single-stranded DNA templates.
2. Annealing: Lowering the temperature to $50\text{ to }55^\circ\text{C}$ to allow sequence-specific synthetic oligonucleotide primers (forward and reverse) to bind to their complementary sites flanking the target region.
3. Extension: Raising the temperature to $72^\circ\text{C}$. A thermostable DNA polymerase (e.g., Taq Polymerase from Thermus aquaticus or Pfu Polymerase from Pyrococcus furiosus) synthesizes complementary strands by adding dNTPs to the $3'\text{-OH}$ ends of the primers.

• RT-PCR (Reverse Transcription PCR): Amplifies RNA targets. Reverse transcriptase first synthesizes single-stranded cDNA from an mRNA template, which is then amplified using standard PCR.
• Real-time qPCR (Quantitative PCR): Quantifies DNA concentration in real-time during amplification using fluorescent dyes (e.g., SYBR Green, which fluoresces only when bound to double-stranded DNA) or fluorogenic probes. Eliminates the need for post-PCR gel electrophoresis.
• Ct Value (Threshold Cycle): The cycle number at which the fluorescence signal crosses a predefined threshold. The Ct value is inversely proportional to the starting amount of target nucleic acid in the sample.

DNA libraries are comprehensive collections of cloned DNA fragments representing an organism's genetic material.

• Genomic DNA Library: Represents the entire genome of an organism, including both coding (exons) and non-coding (introns, promoters, repetitive elements) regions.
  • Construction: Genomic DNA is isolated, partially digested with a restriction enzyme to generate overlapping fragments, ligated into cloning vectors, and transformed into host cells.
• cDNA Library: Represents only the transcribed, active genes (mRNA) of a specific tissue or cell type at a given physiological state. It contains no introns or non-coding genomic sequences.
  • Construction: Total mRNA is isolated, reverse-transcribed into single-stranded cDNA using reverse transcriptase, converted to double-stranded cDNA using DNA Polymerase, ligated into vectors, and cloned into host cells.