Biotech

Biotechnology Overview

  • Definition:

    • Biotechnology is the manipulation of organisms or their components to make useful products.

  • Applications:

    • The applications of DNA technology affect various fields including:

    • Agriculture

    • Criminal law

    • Medical research

DNA Technology

Nucleic Acid Hybridization

  • Definition:

    • Nucleic acid hybridization refers to the base pairing of one strand of nucleic acid to the complementary sequence on another strand.

  • Applications in Research:

    • Researchers exploit complementarity to determine a gene's complete nucleotide sequence, a process known as DNA sequencing.

  • First Automated Procedure:

    • The first automated sequencing method called dideoxy or chain termination sequencing, was developed by Frederick Sanger.

  • Next-Generation Sequencing:

    • Next-generation sequencing techniques are significantly cheaper and faster compared to earlier methods.

Next-Generation Sequencing Techniques

Sequencing by Synthesis

  • Process Overview:

    • In sequencing by synthesis, many DNA fragments are copied.

    • A specific strand of each fragment is immobilized, and the complementary strand is synthesized one nucleotide at a time.

  • Components Involved:

    • Template strand: The original strand used as a template for synthesis.

    • Primer: A short nucleic acid sequence that initiates DNA synthesis.

    • Deoxyribonucleotides: The four building blocks of DNA (dATP, dCTP, dGTP, dTTP).

    • Dideoxyribonucleotides: These are fluorescently tagged and used to terminate DNA strand elongation.

    • Technique Reference: DNA polymerase is used to facilitate strand synthesis.

High-Throughput Technology

  • Description:

    • Thousands or hundreds of thousands of fragments, approximately 300 nucleotides long, can be sequenced in parallel.

    • This exemplifies “high-throughput” technology.

  • Process Steps:

    • Solutions containing each of the four nucleotides are added to all wells sequentially and then washed off.

    • The entire process is then repeated to ensure comprehensive coverage of the sequences.

Detection Mechanism

  • Detection of Nucleotide Addition:

    • If a nucleotide is correctly joined to the growing strand, pyrophosphate (PPi) is released, causing a flash of light that can be recorded.

    • If a nucleotide is not complementary to the next template base, no PPi is released, and hence no flash of light occurs.

DNA Cloning

Definition and Purpose

  • Process:

    • DNA cloning allows scientists to prepare well-defined DNA segments in multiple identical copies.

  • Key Terms:

    • Plasmids: Small circular DNA molecules that replicate separately from bacterial chromosomes.

    • Cloning Vector: A plasmid used specifically to clone a foreign gene.

    • Recombinant DNA: A molecule containing DNA from two different sources, often created by inserting foreign DNA into plasmids.

    • Gene Cloning: The process to produce multiple copies of a single gene.

Applications of Gene Cloning

  • Allows for the expression of proteins from cloned genes for various applications:

    • Production of proteins for medical treatments (e.g., human growth hormone for stunted growth).

    • Alteration of bacteria for bioremediation to clean up toxic waste.

    • Production of proteins for therapeutic use (e.g., blood clot-dissolving proteins for heart attack therapy).

Characteristics of Bacterial Plasmids

  • Advantages:

    • Readily available

    • Easily manipulated

    • Easily introduced into bacterial cells

    • Rapid multiplication once inside bacterial cells

Restriction Enzymes

Definition and Function

  • Definition:

    • Bacterial restriction enzymes cut DNA molecules at specific DNA sequences known as restriction sites.

  • Fragmentation:

    • A restriction enzyme typically makes many cuts, producing restriction fragments.

  • Sticky Ends:

    • The most beneficial restriction enzymes cut DNA in a staggered manner, resulting in fragments with one or more single-stranded ends referred to as sticky ends.

Figures and Mechanisms

Figure 20.5a

  • Shows a bacterial plasmid and restriction site being targeted by a restriction enzyme that cuts sugar-phosphate backbones.

Figure 20.5b

  • Illustrates base pairing of sticky ends producing various combinations when fragments from different DNA molecules are cut by the same restriction enzyme.

Figure 20.5c

  • Displays the sealing of strands by DNA ligase to form recombinant DNA molecules.

Gel Electrophoresis

Purpose and Process

  • Function:

    • Gel electrophoresis is used to check the recombinant plasmid by cutting the products with the same restriction enzyme and separating the resultant fragments based on size.

  • Separation Medium:

    • A gel made of a polymer is employed to separate a mixture of nucleic acids.

Sample Sizes

  • Sample B:

    • Contains fragments of 800 base pairs (bp) and 200 bp long.

  • Sample C:

    • Comprises fragments of 600 bp, 300 bp, and 100 bp long.

Amplifying DNA

The Polymerase Chain Reaction (PCR)

  • Function:

    • PCR generates many copies of a specific target segment of DNA through a three-step cycle involving:

    • Heating

    • Cooling

    • Replication

  • Key Enzyme:

    • Taq polymerase, a heat-stable DNA polymerase, is used in the PCR process.

  • Primer Specificity:

    • PCR uses a pair of primers specific for the sequence intended for amplification.

  • Error Occurrence:

    • PCR amplification may occasionally introduce errors into the amplified strands; therefore, it cannot substitute for gene cloning in cellular processes.

PCR Cycle Breakdown

  • Cycle Steps:

    • Denaturation: Separation of DNA strands.

    • Annealing: Binding of primers to target sequences.

    • Extension: Synthesis of new DNA strands.

  • Outcome of Cycles:

    • Cycle 1 yields 2 molecules.

    • Cycle 2 yields 4 molecules.

    • Cycle 3 yields 8 molecules, with 2 molecules matching the target sequence.