Molecular Techniques Biochemistry and Biotechnology BY Srivastava. (NCBA)

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SKU: 9788173816017

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.