Biotechnology and Microscopy

Define optical microscopy

• AKA light microscopy

• Uses visible light and optical lenses to magnify and view a sample

• Types

  • Fluorescent and compound

Define electron microscopy

• Uses a focuses beam of electrons to magnify and view a sample

• Larger and more expensive, but also more magnified

• Types

  • Scanning electron microscopy (SEM)

  • Transmission Electron Microscopy (TEM)

Describe compound microscopy

• Visible light is focused on a thin slice of the sample

• Produces 2D image

• Uses

  • cells, tissues, organisms

  • staining used

• Disadvantage

  • Can’t view living cells (stain kills)

Describe fluorescence microscopy

• Fluorescent marker to tag certain structures

• Assist in visually locating protein expression within a cell

• Uses

  • Thin slices of living samples

  • Can look at protein expression and specific parts of cell (chromosomes during mitosis)

Describe scanning electron microscopy (SEM)

• Produces a 3D image of a sample’s surface

• Sample must first be dehydrated and coated before viewing

• Uses

  • High resolution with surface level detail (texture, shape, etc)

  • Ideal for viewing external surfaces of cells, tissues, and molecules

Describe Transmission Electron Microscopy (TEM)

• Electron beam passes through a very thin section of sample

• Produces a high magnification 2D image

• Uses

  • Allows for high resolution viewing of internal structures

  • Ideal for viewing internal structure of cells, tissues, and organelles

  • High magnification

• Disadvantages

  • Kills cells

Describe cell fractionation - process

• Homogenization

  • Cell broken apart

  • Cellular homogenate (cell contents without membrane)

• Low speed centrifugation

  • Creates dense pellet layer of nuclei

  • Scrape out pellet layer to study nuclei

• Medium speed centrifugation

  • Remaining homogenate poured out and spun again

  • Mitochondria and chloroplast

• High speed centrifugation

  • Process repeats

  • Leaving smallest components

  • Ribosomes and viruses

Describe vertical gene transfer

• Transfer of genes from one generation to the next

• Examples

  • Sexual/asexual reproduction

  • Mitosis

Describe horizontal gene transfer

• Transfer of genes between different organisms

• Three types

  • Conjugation

    • Transfer of DNA via a bridge

    • Between bacteria via a pilus

  • Transduction

    • DNA introduced into genome via a virus

  • Transformation

    • Absorb DNA from surrounding and incorporate into genome

    • Occurs via heatshocking and electroporating

Describe artificial recombinant technology

• Use restriction enzymes to cut ip specific segments of DNA

• Restriction enzyme cut at sequence-specific sites (recognition sites) —> palindromic sequences

• Restriction enzymes produce:

  • Sticky ends - overhands of nucleotides (mostly used)

  • Blunt ends (no overhanG)

Describe the importance of restriction enzymes

• Sticky ends allows fro new DNA pieces that are cut with the same restriction enzyme to bind

• This creates a DNA molecule from multiple sources

Describe restriction mapping

• Map of restriction enzyme cut-sites within a sequence of DNA

• Useful to know where to cut DNA and what relevant sites are near by

Describe restriction fragment polymorphism (RFLPs)

• Location of restruction site son human DNA will vary between individuals

• DNA fingerprinting

  • Using RFLPs to link an individual to their own DNA in crime scenes or paternity

    • Similar fragments will appear

Describe single nucleotide polymorphims (SNPs)

• Differences in human genome

• May be found near disease associated alleles —> genetic markers

Describe gel electrophoresis

• Used to separate DNA/RNA/proteins based on charge and size

• Samples loaded at the top of the cell next to negative electrode

• Moves to positive end

• Separated based on charge and size

• Smaller = further

• After —> sequenced or probed to identify location of specific sequence

• Probe —> radioactively labelled single strand nuclei acid used to tag a specific sequence

• Proteins

  • Have strong folding structure and can be negative or positive

  • Needs to be treated with SDS to denature and make it into a linear chain

  • Also adds a negative charge coding

Describe nucleic acid hybridization

• DNA or RNA form base pairs with complementary nucleic acids on a different strand

• Used in DNA probing and in-situ hybridization

Describe DNA probing

• DNA probe

  • Denture DNA probe into two single stranded pieces

  • If the DNA has the complementary piece, the DNA probe will bind to it

  • Use detectable label to see if a gene is present

Describe nucleic acid hybridization - in-situ hybridization

• Tests the expression of a specific mRNA using a nucleic acid probe (DNA or RNA)

• Probe labelled with a fluorescent dye

• Probe hybridized with mRNA of interest

• Fluorescent tag allows us to see the mRNA in place on the intact organisms

• Can be visualized within tissues or small embryos

Define DNA sequencing

• Used to determine the # of base pairs in a DNA or RNA molecule and their sequence

• Early method

  • Dideoxy chain termination

• Current

  • Next generation sequencing

Describe dideoxy chain termination sequencing

1. Denature DNA to separate strands

2. Single-stranded DNA is mixed with a primer

   1. Primer provides 3’ OH necessary for DNA polymerase to begin DNA synthesis

3. Samples is incubated

   1. DNA polymerase

   2. dNTPs

   3. ddNTPs (fluorescently tagged)

      1. Lack 3’ OH —> cannot form phosphodiester bond

      2. Deemed as replication terminating nucleotides

4. DNA polymerase adds until the terminating ddNTPs

5. Occurs many times over and we are synthesizing new strands

6. End up with strands of every possible lengths (different nucleotides have different fluorescent colour)

7. Separated via gel electrophoresis from shortest to longest strand

8. Each nucleotide is labelled and ordered digitally via a computer

Describe reverse transcriptase

• Enzyme used to synthesize DNA molecule off an mRNA template

• To create complementary (cDNA)

• Since the template is RNA, there are no introns

• Some viruses (HIV, Hepatitis B) use reverse transcriptase to replicate their genome and proliferate

Why make cDNA?

• Required to create recombinant DNA in bacteria

• Foreign DNA introduced into the bacteria cannot contain introns

• Prokaryotic RNA does not contain introns, so they have no mechanisms in place to remove them

• Allows for gene to be efficiently transcribe and translated

• cDNA is much more stable and long-lasting compared to RNA

Describe the basics of polymerase chain reaction (PCR)

• Important technique for the amplification of DNA

• Necessary ingredients

  • Nucleotides

  • Primers

  • Heat-resistant polymerase (Taq polymerase)

Describe the steps of PCR

• Denature

  • Separate into 2 strands via high temperatures

  • Requires heat resistant polymerase

• Annealing

  • As temperature cools down, primers can attach to individual strands

  • DNA polymerase can only CONTINUE a strand, not make a new one

  • The primer allows for an attachment point

• Elongation

  • The temperature is raised, heat resistant polymerase synthesizes complimentary strands

  • Keep in mind

    • Using a prokaryotic polymerase o human DNA still produces human DNA

    • Prokaryotic polymerase is more stable under heat

• Occurs to both strands at the same time

• PCR is run many times —> exponentially increase

Describe DNA microarray assay

• Monitor the expression of large groups of genes across genome

• See which genes are transcribe in different tissues or at different stages of development

Describe the steps of a DNA microarray assay

• In a bunch of wells, there are short sequences of DNA to see which genes are actively being

• Load cDNA into a pipette and add to the wells (fluorescently labelled)

• No gene present = no hybridization = will not express any fluorescent signals

• Analysis

  • Red —> in cancer cells only

  • Green —> normal cells only

  • Yellow —> both present

  • Grey —> not present

• Used for:

  • Normal vs. cancer cells

  • Different cell types (nerve vs. macrophages)

Describe blotting techniques

• Allow for the indentification of target fragments of DNA, RNA, or proteins

• Types

  • Southern: DNA

  • Northern: RNA

  • Western: Proteins

  • Remember via SNOW DROP

  • To find if a particular gene sequence is presence

Describe the steps of southern blotting

• Extract DNA with gene of interest via restriction enzymes

• Separate DNA fragments by size via gel electrophoresis

• Fragments transferred to nitrocellulose paper and blotting paper is stacked on top of it to create a sucking motion

• Nitrocellulose paper exposed to labelled DNA probe

• Allows DNA fragments to be visualized by hybridizing if present

Define immunofluorescent staining

• Staining technique

  • Allows for the visual identification of proteins

List the steps of immunofluorescent staining

1. Addition of primary antibody to bind to a specific protein

2. Addition of secondary antibody which contains fluorescent tag and binds primary antibody

3. Visualization of protein of interest using microscopy, the fluorescent ga can be visually located to detect the protein of interest

Describe in-vivo mutagenesis

• Helps determine the function of the gene by seeing what goes wrong without the functional copy of that gene

List the steps of in-vivo mutagenesis

1. Introduce a specific mutation into a gene to disrupt its functions

2. Observe for any phenotypic differences

3. Differences may be a function of a missing normal protein

4. Frequently used example

   1. Knock out mice

In-vitro mutagenesis —> similar, but occurs OUTSIDE of a living organism (cells in a culture)

Describe genome annotation

• Analyzing genomic sequences to identify the protein-coding regions and their functions

• Utilizes computer databases to compare known sequences

• Identifying functioning and non-functioning elements of a genome

Describe gene therapy

• Introduction of genes into an afflicted individual for therapeutic purposes

  • Using a retroviral vector to insert genome material into chromosomal DNA

  • Non functional DNA segments have been replaced with functional ones

Describe transgenic animals

• Animals which have a gene introduced from the genome of another individual

• Transgenic mice —> implanted with a gene from a jellyfish that expresses a green fluorescent protein

Define genomic library

• Collection of cloned DNA pieces from a genome

• Library can be screened to locate a gene of interest

List the steps of creating a genomic library

1. Isolate genome of interest (extract DNA)

2. Cute genomic with restriction enzymes

3. Cut plasmid with same restriction enzymes (small, double stranded, circular DNA)

4. Ligate genes into plasmid

5. Insertion of plasmid into bacteria (via transformation)

   1. Called making cell “compotent”

   2. Using heat shock or electroporation

6. Allow bacteria to multiply to replicate genome

7. DNA isolation

Describe heat shock and electroporation

• Electroporation

  • Brief electrical impulse

  • Creates temporary pores in plasma membrane

• Heat shock

  • Temperature increased then rapidly cooled

  • Increases membrane permeability

How do we know if teh bacetria were transformed?

• Plasmid used in genomic libraries contain antibiotic resistance genes

• Antibody resistance test

  • Helps to verify which bacteria were successful in accepting the plasmid

• Treat all bacteria with an antibiotic

• Bacteria who didn’t survive indicate a lack of the antibiotic resistance gene

  • Unable to take up the plasmid

• Bacteria who survive prove they are resistant to the antibiotic

  • Deemed ‘recombinant bacteria’ due to their successful uptake of the plasmid

List the steps of reproductive cloning

1. Isolate donor cell with nucleus

2. Isolate unfertilized enucleated egg from donor

3. Transplant nucleus into enucleated egg (electroporation)

4. Embryo formation

5. Transfer embryo into surrogate mother

6. Deliver baby clone

   1. Complete clone to sheep that was the nucleus donor (somatic cell’s nucleus)

Describe Pasteurs Swan Neck Flask Experiment

• Proved spontaneous life-generation was invalid

• Life cannot be created from non-life

Describe Griffith’s experiment

• Used 2 strains of bacteria that he injected into mice

  • Rough (R) strain —> lacked protective capsule and was therefore non-virulent (pathogenic)

  • Mic’s immune system killed the bad cells

  • Vector lives

• Smooth (S) strain —> protective capsule shielding it from the immune system

  • Vector dies

• Both R and S

  • Vector dies

  • The other plasma enters bacteria cells via transformations

Describe the Avery-McLeod-McCarty experiment

1. Separate addition of various digestive enzymes to the heat killed S’ bacteria

2. Digestive enzymes included: DNases, proteases, and lipases

3. Most of these mixtures had no effect

4. HOWEVER, when DNase was added to the heat-killed S’ bacteria, the R cells were not transformed

   1. Any DNA in the tube was killed, which is needed for R’ bacteria to become virulent

   2. R bacteria never gained ability to produce the protective capsule

   3. Proteases + S’ bacteria would result in protein breakdown and unaffected DNA

      1. Bacteria would still be transformed

Describe Hershey and Chase experiment

• Showed that DNA, not proteins, was the genetic material of Phage T2 (a virus)

  • Virus that affects bacteria

  • Placed a radioactive label in DNA of the virus

    • Placed a radioactive label on sulfur in the protein of the virus

    • Sulfur is specific to proteins

    • None of radioactive labelled sulfur was identified in bacteria, but phosphorus was

    • Confirmed DNA is the genetic material of a virus

Describe Meselson and Stahl’s experiment

• proved semiconservative replication model was the valid DNA model

• 1 Parent strand and 1 new strand

1. Grew E. Coli in medium with nucleotides

2. Bacteria transferred to medium with 14N

3. 15N bacteria replicated in new medium

4. Replication continued for another round

Describe Gurdon’s Nuclear Transfer Experiment

• Proved that fully differentiated cells do not lose their genetic information - and still retain full genome

• Placed a nucleus from a differentiated frog cell into an enucleated egg cell —> gave rise to a new frog