Biotechnology & Microscopy Notes

Biological Theories

• Biological Scale: Covers the range of sizes and complexity in biological systems.

• Cell Theory:

  • All living organisms are composed of one or more cells.

  • The cell is the basic unit of structure, function, and organization in all organisms.

  • All cells come from preexisting, living cells.

  • Cells carry hereditary information.

• RNA World Hypothesis:

  • Self-replicating RNA molecules were precursors to life.

  • RNA stores genetic information like DNA.

  • RNA can catalyze chemical reactions like enzymes.

• Central Dogma of Biology:

  • The general flow of genetic information is from DNA to RNA to protein.

Biological Technology

• Microscope Techniques:

  • Light Microscope: Uses visible light to view a thin sample.

  • Scanning Electron Microscope: Used to view the surface of 3D objects with high resolution.

  • Transmission Electron Microscope: Used to view thin cross-sections and internal structures within samples at very high magnification.

  • Confocal Laser Scanning Microscope/Fluorescence: Can observe specific parts of a cell using fluorescent tagging antibodies (e.g., used to view chromosomes during mitosis).

• Polymerase Chain Reaction (PCR):

  • PCR creates a large amount of DNA by amplifying a DNA sample through the following steps:

    1. Denaturation: High heat separates double-stranded DNA.

    2. Annealing: Sample is cooled so primers attach to separated strands.

    3. Elongation: Polymerase synthesizes new strands.

  • Cycle repeats to increase the amount of DNA exponentially.

• Reverse Transcriptase:

  • Used to synthesize DNA from an RNA template.

  • In some lab procedures, it is used to create complementary DNA (cDNA) off an mRNA template. cDNA lacks introns.

  • Naturally used by viruses.

• Centrifugation:

  • Technique to separate a liquefied sample into its different components by spinning it rapidly.

  • Largest/most dense separate first, forming a pellet at the bottom of the sample.

  • Cell Fractionation (largest to smallest): Nuclei > mitochondria/chloroplast > ribosomes.

• DNA Sequencing:

  • Used to determine the sequence of base pairs in a DNA or RNA molecule.

  • Example: Dideoxy Chain Termination is based on the principle that during DNA synthesis, the addition of a nucleotide requires a free OH group on the 3' carbon of the sugar of the last nucleotide of the growing DNA strand.

• Blotting Techniques:

  • Blotting techniques are used for identifying specific fragments of DNA, RNA, or protein.

  • 3 types of Blotting:

    • Southern - DNA

    • Northern - RNA

    • Western - Protein

  • Blotting Methodology:

    1. Electrophoresis is used to separate the sample.

    2. The sample is transferred to a nitrocellulose gel.

    3. A probe is added to hybridize and mark the target fragment (Hybridization is when nucleic acids form complementary base pairs with nucleic acids of a different strand).

• Gel Electrophoresis:

  • Used to separate DNA molecules by size and charge: the smaller the molecule, the farther it travels down the gel.

  • After separating the DNA sample, it can be sequenced or probed to find the location of a specific sequence.

• Microarray Assays:

  • Used to monitor the expression of large groups of genes across a genome.

• Recombinant DNA & Gene Libraries:

  • Recombinant DNA contains segments from multiple sources. The process to making and using recombinant DNA includes:

    1. Using restriction endonucleases (restriction enzymes) to cut specific segments of DNA called restriction sites. These enzymes create sticky ends, which allow new DNA pieces to bind.

    2. DNA ligase connects the different fragments together.

    3. A vector can then be used to transfer foreign DNA into another cell. Examples of vectors include plasmids and bacteriophages.

  • Example of recombinant DNA in bacterial cloning:

    1. A restriction enzyme is applied to both the bacterial plasmid and the foreign DNA to create the same sticky ends.

    2. DNA ligase attaches the fragments to create a plasmid with new DNA.

    3. The plasmid is introduced into bacteria using transformation.

    4. Bacteria can be grown to produce a product or form a colony. To ensure that the bacteria has included the plasmid, a gene for antibiotic resistance is added to the plasmid. Bacteria without the plasmid will perish.

  • Recombinant DNA is vital for creating gene libraries, which are collections of DNA pieces from a genome.