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Medical Biochemistry and Biotechnology BY Mohammed Amanullah, (NCBA)
Original price was: ₹585.00.₹470.00Current price is: ₹470.00.
1. Nucleic Acid Extraction & Purification
Before any molecular manipulation can occur, pure genetic material must be isolated from cellular matrices.
- Cell Lysis: Mechanical disruption or chemical lysis using detergents like Sodium Dodecyl Sulfate (SDS) or Sarkosyl to break open lipid bilayers.
- Enzymatic Digestion: Utilizing Proteinase K to degrade contaminating histones and cellular proteins.
- Separation: Phenol-chloroform extraction or solid-phase silica spin columns to bind and isolate nucleic acids from polysaccharides and lipids.
- Quantification: Measuring concentrations using UV spectrophotometry at a 260 nm wavelength.
2. Amplification & Quantitation Techniques
These methods copy and track small quantities of specific nucleic acid targets.
- Standard Polymerase Chain Reaction (PCR): Thermally cycles through denaturation (~95°C), primer annealing (~55°C), and extension (~72°C) via heat-stable Taq polymerase to exponentially amplify DNA fragments.
- Reverse Transcription PCR (RT-PCR): Uses reverse transcriptase to convert unstable viral or cellular mRNA into complementary DNA (cDNA) before standard amplification.
- Quantitative Real-Time PCR (qPCR): Incorporates fluorescent dyes (e.g., SYBR Green) or TaqMan probes to monitor DNA copy accumulation in real-time, functioning as a primary tool for measuring gene expression.
3. Separation & Blotting (Macromolecular Analysis)
Once isolated or amplified, fragments are sorted by physical traits and cross-referenced with target probes.
- Gel Electrophoresis: Agarose gels separate large DNA/RNA fragments based on size as an electric current pulls negatively charged backbones toward a positive anode. Polyacrylamide Gel Electrophoresis (PAGE) is reserved for proteins or smaller oligonucleotides.
- SDS-PAGE: Denatures proteins and coats them with a uniform negative charge, separating them strictly by molecular weight.
- Blotting Systems: Capillary or vacuum transfer of resolved bands from unstable gels onto durable nitrocellulose/PVDF membranes.
- Southern Blot: Probes immobilized DNA targets.
- Northern Blot: Probes immobilized RNA target profiles.
- Western Blot (Immunoblotting): Uses primary and secondary enzyme-linked antibodies to target proteins.
4. Recombinant DNA Technology & Cloning
Genetic engineering relies on cutting, pasting, and propagating sequences inside foreign host models.
- Restriction Digests: Using bacterial restriction endonucleases to cleave specific, palindromic target DNA sequences, leaving clean sticky or blunt ends.
- Ligation: Utilizing DNA Ligase to establish covalent phosphodiester bonds, pasting target genes into vector backbones.
- Cloning Vectors: Engineering circular plasmids with origin of replication zones (ori), multiple cloning sites (MCS), and antibiotic selection markers.
- Transformation & Transfection: Forcing recombinant plasmids into competent bacteria via heat-shock/electroporation (Transformation), or introducing foreign DNA into eukaryotic host cells using chemical liposomes or calcium phosphate (Transfection).
5. High-Throughput Sequencing & Omics
Modern biotechnology workflows process thousands of global molecular targets simultaneously.
- Sanger Sequencing: Chain-termination method utilizing dideoxynucleotides (ddNTPs) to decode exact, individual single-strand sequences.
- Next-Generation Sequencing (NGS): Massively parallel sequencing strategies that process entire complex genomes or transcriptomes in a single run.
- DNA Microarrays: Solid surface chips hosting hundreds of microscopic DNA spots to analyze global comparative gene expression or profile genetic polymorphisms.
- CRISPR-Cas9 Editing: RNA-guided endonuclease system creating precise, targeted double-strand breaks to rapidly knock out or knock in genes.
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