Biotechnology and Microscopy

Optical Microscopy

Utilizes visible light and optical lenses to magnify and view a sample

Electron Microscopy

Utilizes a focused beam of electrons to magnify and view samples

Compound Microscope

Visible light is focused on a thin slice of the sample → produces a 2D image

Compound Microscope Uses

• Observation of a wide variety of cells, tissues, and organisms

• Staining can be used for enhanced viewing

Compound Microscope Disadvantages

• Limited view of living samples (only a single cell layer)

• thicker samples require staining which kills the sample

Fluorescence Microscopy

• Use of a fluorescent marker to tag certain structures

• Assist in visually locating protein expression within a cell

Fluorescence Microscopy Uses

• View thin slices of living samples

• can look at protein expression and specific parts of cell (e.g., chromosomes during mitosis)

Scanning Electron Microscopy

• produces 3D image of a sample’s surface

• sample must first be dehydrated and coated before viewing

Scanning Electron Microscopy Uses

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

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

Transmission Electron Microscopy

Electron beam passes through a very thin section of sample → produces a high magnification 2D image of sample

Transmission Electron Microscopy uses

• allows for high resolution viewing of internal structures

• ideal for viewing internal structures of cells, tissues and organelles

• highest magnification of all microscopes

Cell Fractionation

uses differential centrifugation (spinning at high speeds) to separate a cell’s contents based on density and size

Cell Fractionation - Process

• Homogenization

• Low-speed centrifugation

• Medium-speed centrifugation

• High-speed centrifugation

High-speed centrifugation

Process repeats → leaving smallest cell components → ribosomes + viruses

Medium-speed centrifugation

Remaining homogenate poured our and spun again → creates layer of mitochondria and chloroplast

Low-speed centrifugation

Cell homogenate spun → creates dense pellet layer of nuclei

Homogenization

Cells broken apart → cellular homogenate (cell contents without membrane)

most dense layer of cell

the nuclei

middle dense layer of cell

mitochondria / chloroplasts

least dense layer of cells

ribosomes/viruses

Vertical Gene Transfer

Transfer of genes from one generation to the next i.e., Sexual/asexual reproduction/mitosis

Horizontal Gene Transfer

transfer of genes between different organisms

Conjugation

transfer of DNA from donor cell to a recipient cell

Transduction

DNA is introduced into genome by a virus

Transformation

absorb DNA from surrounding and incorporate into genome

Recombinant DNA

DNA containing different segments from multiple sources

• segments can then be transferred via: bacterial conjugation, viral transduction, transformation, and artificial recombinant technology

You use what to cut up DNA?

restriction enzymes - cut at sequence-specific sites (recognition sites) → palindromic sequences

Palindromic Sequences

Nucleic acid sequences that read the same in both directions

• 5’ → 3’ = AGTACT

• 3’ → 5’ = TCATGA

Cut Ends

Restriction enzyme cuts can separate nucleotides in different ways

• sticky ends (overhangs of nucleotides)

• Blunt ends (no overhang)

Restriction Mapping

creating a map of known restriction enzyme cut-sites within a sequence of DNA

Restriction Fragment Polymorphisms (RFLPs)

The location of restriction sites on human DNA will vary between individuals, applying restriction enzymes to human DNA will create unique fragmetns of different sizes

DNA Fingerprinting

Using RFLP’s to link an individual to their own DNA in crimes scenes or paternity lawsuit

Single Nucleotide Polymorphisms (SNPs)

single nucleotide differences in the human genome → one in roughly every 1000-2000 nucleotides

• may be found near disease-associated alleles → used as genetic markers for certain diseases

Gel Electrophoresis

can be used to separate DNA/RNA/proteins based on charge and size

Gel Electrophoresis - Process 1

Different DNA molecules are added to an agarose gel under an electric field

Gel Electrophoresis - Process 2

Negatively charged DNA moves away from negative end to positive end

• shorter DNA molecules move further that larger DNA molecules

Gel Electrophoresis - Process 3

After electrophoresis → DNA can be sequenced (or probed) to identify the location of the specific sequence

Gel Electrophoresis - Process 4

DNA probe → radioactively labelled single srand nucleic acid used to tag a specific sequence

Gel electrophoresis for proteins is similar, but with a key difference…

addition of SDS

SDS is added prior to loading which….

denatures the protein into a linear polypeptide chain

→ resulting polypeptide is uniformly negatively charged

Nucleic Acid Hybridization

• Nucleic acids of one strand (DNA or RNA) form base pairs with complementary nucleic acids on a different strand

• Used in many techniques → DNA probes, in-situ hibridization

In-situe hybridization

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

IN-situe hybridization - process 1

probe labelled with a flourescent dye

IN-situe hybridization - process 2

Probe hybridizes with mRNA of interest

IN-situe hybridization - process 3

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

• can be visualized within tissues or small embryos

DNA Sequencing

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

• early method of DNA sequence → Dideoxy chain termination

• Current → Next generation sequencing

What do you need for Dideoxy Chain Termination Sequencing

• DNA

• DNA polymerase

• Primer

• DNA (dNTP)

• fluorescent tagged (ddNTP)

Dideoxy Chain Termination Sequencing Process 1

Desired DNA strand is denatured into a single-strand form

Dideoxy Chain Termination Sequencing Process 2

Single-stranded DNA is mixed with a primer

Dideoxy Chain Termination Sequencing Process 3

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

Dideoxy Chain Termination Sequencing Process

sample of ssDNA + primer is incubated with:

• DNA polymerase

• dNTPs

• ddNTPs → fluorescently tagged

ddNTP

• lack an OH group on 3’ carbon

• they can be added because they have the 5’ phosphate end, but the cannot add another one

• termination occurs once you add the ddNTP

Labelled strands are separated via…

capillary gel electrophoresis form shortest to longest strand

• each nucleotide from target strand is labelled and ordered

Sequencing - technology detects the order of fluorescent colors, which indicates…

the exact sequence of the original target strand

Reverse Transcriptase

Enzyme used to synthesize DNA molecules off an mRNA template

Reverse transcriptase is used in lab procedures to…

create complementary DNA (cDNA) off an mRNA template

• NO INTRONS

Some viruses use reverse transcriptase to…

replicate their genome and proliferate in the host (HIV and Hepatitis B, Retroviruses)

Prokaryotic RNA does not contain introns, so…

they have no mechanism in place to remove them

cDNA created from the mRNA template doesn’t contain introns found in the …

original DNA template (because the mRNA template has already edited these portions out) which allows for desired gene to be efficiently transcribed and translated after insertion

Why wouldn’t you just insert the mRNA directly into the bacteria for translation?

RNA is inherently unstable and short-lived - cDNA is much more stable and long lasting

Polymerase Chain Reaction

Important technique for the amplification of DNA

• Necessary ingredients: nucleotides, primers, and heat-resistant polymerase

PCR Steps: 1

Denaturation

• double stranded DNA molecule is heated to a high temperature to separate into strands

• use of high temperatures in PCR process required a heat-resistant polymerase (avoids denaturation)

PCR Steps: 2

Annealing

• as the temperature cools down, the primers can attach to separate strands

PCR Steps: 3

Elongation

• The temperature is raised -. heat-resistant polymerase synthesizes complementary strands

• using prokaryotic polymerase on human DNA still produces human DNA

• prokaryotic polymerase is more stable under heat

DNA Microarray Assay

Moniter the expression of large groups of genes across the entire genome

What is DNA Microarray Assay useful for?

for seeing which genes are transcribed in different tissues or at different stages of development

DNA Microarray Assay Process: 1

Tiny amounts of ssDNA fragments representing different genes are fixed to a glass slide in a grid of dots (array)

DNA Microarray Assay Process: 2

mRNAs are isolated from a cell → reverse transcriptase is used to make cDNA

DNA Microarray Assay Process: 3

cDNAs are labelled fluorescently and then allowed to hybridize to DNA microarray

DNA Microarray Assay Process: 4

Differentiate samples/tissues with different color labels

• Lit up wells → expressed gene

• Compare gene expression in: Norma vs cancer cells and then different types of cells

Southern Blotting

used to identify DNA fragments

northern blotting

used to identify RNA molecules

Western blotting

used to identify proteins

Southern Blotting: Step 1

Extract DNA with Gene of Interest

• DNA is extracted from a biological sample (blood or tissue)

• cut into fragments via restriction enzymes

Southern Blotting: Step 2

Separate DNA fragments by size

• via electrophoresis

Southern Blotting: Step 3

Fragments transferred to nitrocellulose paper

• first palced on top of gel, then blotting paper is placed on top

Southern Blotting: Step 4

Nitrocellulose Paper Exposed to Labelled DNA Probe

• labelled with a radioactive, fluorescent, or chemical tag

Southern Blotting: Step 5

Allows DNA fragments to be visualized

Immunofluorescent Staining

Staining technique

Immunofluorescent Staining Step: 1

Addition of primary antibody

• binds to protein of interest

Immunofluorescent Staining Step: 2

Addition of Secondary antibody

• contains fluorescent tag and binds to first antibody

Immunofluorescent Staining Step:

Visualization of Protein of Interest

• fluorescent tag can be visually located to detect the protein of interest

In-vivo Mutagenesis

Helps determine the function of a gene

In-vivo Mutagenesis Step: 1

Introduces specific mutations into a gene

In-vivo Mutagenesis Step: 2

Observe for any phenotypic differences

In-vivo Mutagenesis Step: 3

Differences may be a function of a missing normal protein

In-vitro mutagenesis, only studies the effects of the…

mutation outside of a living organism (e.g., cells in a culture)

Genome Annotation

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

• utilizes computer data bases to compare known sequences

Gene Therapy

Introduction of genes into an afflicted individual for therapeutic purposes

• e.g., using a retroviral vector to insert genome material into chromosomal DNA

Transgenic Animals

Animals which have a gene introduced from the genome of another individual - often another species

Transgenic mice → implanted with a gene from a jellyfish that expresses a gree fluorescent protein, what does this do?

causes the mouse to glow in the dark

Genomic Library

Collection of cloned DNA pieces from a genome

• can be screened to locate a gene of interest

Formation of Genomic Library: Step 1

Isolate Genome of Interest

Formation of Genomic Library: Step 2

Cut Genome with Restriction Enzymes

• site is cut → leaving sticky ends

Formation of Genomic Library: Step 3

Cut Plasmid with Same Restriction Enzymes

• Plasmids → circular dsDNA with restriction enzyme cut-sites

• Also cut → leaving compatible sticky ends

• Plasmid is designed to include an antibiotic resistance gene

Formation of Genomic Library: Step 4

Ligate Genes into Plasmid

• sticky ends of new DNA with bind sticky ends of plasmid

• both will be sealed using DNA ligase

Formation of Genomic Library: Step 5

Insertion of Plasmid into Bacteria (via transformation)

• Bacteria must be made ‘competent’ to take up the plasmid

• can be done via heat shock + CaCl₂ or electroporation

Formation of Genomic Library: Step 6

Allow Bacteria to Multiply to Replicate Genome

• undergo repeated cell division to produce a population of genetically identical cells

• recombinant plasmid, foreign DNA, and other genes are cloned as well

Formation of Genomic Library: Step 7

DNA Isolation as Needed

• the genomic DNA library can now be isolated again for use or experimentation

Electroporation

brief electrical impulse → creates temporary pores in plasma membrane