DAT Biology: Microscopy and Lab Techniques

Microscopy

magnifies the image of a given specimen using preparative techniques to ease viewing and interpretation of a specimen

Fixation

Sticking/securing cells to a slide, preserving them in as close to a lifelike state as possible

Staining

Application of stains/dyes to a specimen to add color/contrast

Allows different structures to become more easily distinguished, but often kills the specimen

Optical Microscopy

Cells viewed directly, thin specimen are illuminated via a light source, while the lenses magnify the resulting image

Living cells can be viewed this way

Stereo Microscopes

Aka dissection microscopes—low magnification is used to view the surface of a specimen

Compound Microscope

Multiple lenses are used to provide variable, adjustable magnification

Viewing of simple, one-cell thick samples requiring fixatino and staining

Poor image contrast

Bright Field Microscopes

Compound microscope with a bright, white light

Phase Contrast Microscope

View thin samples of live, unstained cells with high image contrast

Do NOT need to be stained, embedded, or coated because phase contrast microscopy naturally enhances contrast

Fluorescence Microscopy

View live cells that are tagged with fluorophores, cellular components are visible with this method, allows you to view live specimen

Fluorophores

Fluorescent chemical markers used to tag target structures

Fluorescence Recovery After Photobleaching (FRAP)

Quantitatively measures biomolecule dynamic movement of proteins in a life cell

Electron Microscopy

Cells viewed indirectly through a screen

Electrons are fired at sample, bounce off sample causing them to pass through magnetic field

Smaller objects can be viewed, specimen must be stained with a metal coating killing them

Scanning Electron Microscope (SEM)

High resolution 3D images of hte surface of a dehydrated sample

Transmission Electron Microscope (TEM)

High resolution 2D images of the internal structures of a sample

Bacterial Growth Curve

Graphic depiction of the phasic growth and death of bacteria during cell culturing

X-Axis: Time, Y-Axis: Number of bacterial cells on a log scale

4 Phases: lag phase, exponential phase (log phase), stationary phase, death phase

Lag Phase

Bacterial proliferation is stagnant as cells initially adapt to the new environment (culture medium), absence of growth (characterized not by linear, but no growth)

Growth Rate = Death Rate

Exponential Log Phase

A period of exponential cell growth

Growth Rate > Death Rate

Stationary Phase

A second period of stagnant proliferation, but total population is much higher than that of lag phase

Growth Rate = Death Rate

Death Phase

Rapid decline in bacterial population size caused by lack of resources after log and stationary phases

Death Rate > Growth Rate

Differential Centrifugation

Separating solutes in a given solvent based on size/density

Several rounds of centrifugation, with each round increasing centrifugal forces, where each step selectively pellets components within a specific size/density range (most dense centrifuges first)

Allows fractionation of cell organelles and macromolecules

Pellet

Components which have sedimented to the bottom of the tube

Karyotyping

Observing chromosomes under a microscope to identify potential chromosomal abnormalities during metaphase

Example: trisomy 21 / down syndrome

CRISPR

Technology allowing the editing of specific genomic regions where target sequences may be inserted or deleted

Uses: gene therapy

DNA Fingerprinting

Identification technique which relies on inherent uniqueness of each human genome

Specific regions of noncoding DNA are fragmented with restriction enzymes and analyzed

Uses: paternity testing and forensic identification

DNA Sequencing

Determining the order of nucleotides in a given DNA sequence

Sanger Sequencing

Using PCR to make DNA copies out of deoxynucleotides (dNTPs) and dideoxynucleotides (ddNTPs), which create a variety of fragment lengths, allowing the sequence to be determined (each fragment only tells you the ending letter, so a 4 letter sequence only tells you the 4th letter)

ddNTP

Lack the 3’ OH necessary to create phosphodiester bonds and continue the DNA sequence, ending elongation once inserted so it is readable

Polymerase Chain Reaction (PCR)

Automated process allowing for the creation of 1+ billion copies of a specific DNA fragment

Occurs in 3 steps: Denaturation, annealing, elongation

PCR Denaturation

94-95 degrees C

Intense heating separates the DNA double strands into single strands

PCR Primer Annealing

55 degrees C

Sequences are cooled, allowing DNA primers to hybridize with the single strands

PCR Elongation

72 degrees C

Moderate temperature elevation encourages Taq polymerase activity, adding nucleotides to the 3’ ends of the strands to finish the recreation of the DNA

Bacterial Cloning

Eukaryotic Gene Products are cloned using prokaryotic cells

Uses: medicine production

Gene Therapy

Medical Treatment where target genes are inserted into patient cells, often via viruses, or non-viral vectors like CRISPR and naked plasmid DNA

uses: treatment of diseases with genetic involvement

Enzyme-Linked Immunosorbent Assay (ELISA)

Determines if a person possesses a specific antigen by placing antibodies on a microtiter plate with a sample from the individual, and if the individual possesses the antigens of interest, a color change occurs

Use: disease diagnosis (HIV)

Pulse Chase Experiments

Observing protein movement through a cell

Uses: study gene expression and protein fates (synthesis, movement, degradation) within a cell

Pulse Phase

Amino acids are radioactively labeled and then incorporated into proteins, and radioactive proteins are tracked via simple staining

Chase Phase

Prevention of radioactively labeled protein production

Gel Electorphoresis

Separate DNA fragments/proteins by size/relative charge by placing samples into wells within an agarose gel—smaller and more negative fragments travel further from the top of the gel, toward the positive anode

Uses: genotyping, fingerprinting, diagnostic testing

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

Used in protein gel electrophoresis, proteins fragments are separated out based on density and size for analysis

Uses: assessing protein size and purity

Sodium Dodecyl Sulfate (SDS)

A strong detergent denaturing proteins and giving them a negative charge

Makes proteins uniform for observation

Transformation

process where competent bacterial cells take up plasmic vectors from the environment

Transduction

Process by which foreign DNA is introduced into a cell by a virus (needs a virus bacteriophage to move DNA)

Exonuclease

Nucleotide cleaving enzyme cleaving nucleotides from the ends of a polynucleotide chain and produces sticky ends

Endonuclease

Nucleotide cleaving enzyme cleaving nucleotides from the inside of a polynucleotide chain and producing blunt or sticky ends

Sticky Ends

Single-stranded cuts in a DNA (only one complementary strand is affected), overhangs, including unpaired nucleotides

Blunt Ends

Double-stranded cut in a DNA (both strands synchronously cut), symmetrical cuts, no unpaired nucleotides

Restriction Enzymes

Special endonucleases that mostly cut DNA at palindromic sequences to create blunt or sticky ends

Palindromic Sequence

Short regions where the sequence reads the same in the 5’→3’ direction on both strands

Recombinant DNA

DNA produced when DNA fragments from different sources are joined together at blunt and/or sticky ends

Southern Blotting

Technique used to identify fragments of a known DNA sequence within a large population of DNA by binding exclusively to those matching fragments/complementary strands from a DNA crowd

DNA Probes

Single-stranded segments of DNA labeled with a marker that match and bind to a specific target gene sequence

Northern Blotting

Identify known fragments of RNA using an RNA probe, conducted similarly to the Southern Blot but using RNA

Western Blotting

Quantifies the amount of a target protein in a sample

Uses the SDS page gel to separate protein parts by size, and then transfers them to a membrane where they are treated with primary antibodies, which bind to the target protein, and secondary antibodies, which bind to both an indicator and the primary antibody

Genomics

Study of all genes present in an organism’s genome and how they interact

Gene Annotation

Process of identifying the location of genes and coding regions within a genome and determining each of their functions

Requires a genomic library and DNA microarrays

Genomic Library

Stores the DNA of an organism’s genome

Plasmid

circular, extra chromosomal DNA structures found in prokaryotic organisms

DNA Microarray

Physical means used to determine if a known gene is present within an organism via thousands of DNA probes, allowing researchers to see which genes are expressed and at what levels

Reverse Transcriptase

Enzyme used on processed mRNA to make cDNA