Lectures 19, 20, 21, 22

Question 1

What is western blotting (immunoblotting)?

Answer 1

Method to look for a protein of interest from a gel using antibodies against the protein
Polyacrylamide protein gel is first run (SDS, native 2D)

The gel is transferred to a membrane with a high affinity for protein, the membrane is treated to bind the antibody of interest and visualized

Used to detect protein from sample, expression profiles of a protein in tissue, verification of transgenic organisms, detect infections

Slide 6 L19

Question 2

How is blotting blocking and detecting of antibodies done?

Answer 2

The membrane has a high affinity for protein, so all empty spots must be filled before antibodies can be added (BSA or skim milk powder are used)
Once blocked the 1st degree antibody is added to the blog and allowed to bind
A 2nd degree antibody may then be used to detect the presence of the 1st degree antibody
(Secondary antibodies are antibodies that bind to other antibodies)
Slide 4 L19

Question 3

What are the 4 types of antibody labelling?

Isotope, enzyme, fluorescent, metal

Answer 3

Isotope labelling- I or S are attached to the protein, as isotope decays the relaxed electrons expose X Ray film layered over the blot
Enzyme labelling- common enzyme like horseradish peroxidase (HRP) or alkaline phosphotase (AP) [AP used to decolour]
Enzyme catalyzes reaction to give coloured substance or chemilumescence (detectable light)
Fluorescent labelling- dye attached that absorbs one colour of light and gives off another (can make antibodies glow)
Metal labelling- gold, tungsten, neodymium, and more (visible with electron microscopy)

Question 4

How is HRP + chemiluminescence used for crime scenes?

Answer 4

Spray luminol on crime scenes, glows where blood is since our blood has peroxidase to react with luminol that produces chemiluminescence

Slides 7-8 L19

Question 5

How is an ELISA test and western blot used to confirm HIV?

Answer 5

If an ELISA test shows a positive result the patients serum is used to probe a blot if HIV proteins
ELISA test is a fast western blot (has a lot of false positives, saying someone who doesn’t have HIV, does)
HIV proteins on gel, primary antibody is IgG in your blood, secondary antibody is IgG goat-anti-human HRP
Slide 14 L19
If you have two of three red box regions (any antibodies sticking to them) you are positive for HIV

Question 6

What are ELISA tests?

Answer 6

Steps similar to western blots, but no protein separation
Samples are placed in wells, or spotted into membranes and protein absorbs to surface
Blocking proteins are added, washed away
Detection antibodies added, washed away
Secondary antibody with linked enzyme added, washed away
Chromogenjc reagent added, samples quantified
Pregnancy tests are an example
Slides 15-16 L19

Question 7

How do strip sandwich ELISA tests work?

Answer 7

Used to detect pathogens, blood, drugs, genetically modified foods and more

Mobile enzyme-linked monoclonal mouse anti-HCG at start
Position 2 is immobilized polyclonal mouse anti-HCG (pregnant or not)
Position 3 is immobilized polyclonal anti-mouse Ab (test worked or not) positive control, something should always bind there

Slide 17 L19

Question 8

What are agglutination tests?

Answer 8

Used to test cell specimens (bacterial cells, blood cells)
Emulsion of cells is added to an antibody mixture and rocked back and forth

2 ways to test:
Unknown cells with specific antibody
Known cells with unknown serum

Slide 18 L19

Question 9

What is a diffusion assay?

Aka precipitin reaction or ouchterlony assay

Answer 9

A ring of Wells is cut into an agar plate around a central well (antibodies/serum in center, protein sample in centre)
Ab & Ag diffuse through the agar and will form a visible precipitate between the wells of they are compatible
The continuity of the line between sample can indicate shared antigens
Slide 19 L19 - slide 4-5 L20

Question 10

What is a non identity, partial identity, and identity in the ouchterlony double diffusion test?

Answer 10

Slide 4 L20
Non-identity: intersect lines (has antigens for both)
Partial identity- curved spur since there is something the same between two non centres, extended line means that home has something extra compared to other hole
Identity- whatever is bound is the same

Question 11

Do some peoples bodies reject IgA?

Answer 11

Yes some people make antibodies against IgA which results in severe IgA deficiency
1 in 546 people have IgA deficiency and 2/3 of those don’t have anti-IgA and 1/3 have anti-IgA

Slides 5-6 L20

Question 12

Study the hypothetical flu ouchterlony test on slide 7 L20

Answer 12

Okay

Question 13

What is fluorescence microscopy?

Answer 13

Cultured cells or tissue sections are placed in slides
Cells are fixed using paraformaldehyde or methanol/acetone & rinsed
Cells are permeabilized using dilute detergent (detergent strips off membrane so antibodies can get in)
Cells are blocked to reduce nonspecific binding
Primary antibody is added/washed
Secondary fluorescent antibody is added/washed

Question 14

What is standard, confocal, and two-photon fluorescent microscopy?

Answer 14

Standard- whole image is illuminated and out of focus light makes the in focus image fuzzy (cheap but dizziness and photo damage)
Typical microscope
Confocal- whole image is illuminated and out of focus light is blocked, strip out focus light so only light within range with layer of cells is used (good resolution but photodamage)
Two-photon- less energetic light is used (infrared) and 2 photons must hit the dye simultaneously to excite it (lower photodamage and better tissue penetration but most costly, inferior resolution to confocal) can’t see infrared so if it bounces off slide it can blind you
Slides 9-13 L20

Question 15

What is the review of DNA?

How it’s held together, backbone, 4 bases

Answer 15

Double stranded polymer of deoxyribonucleotides
Held together by hydrogen binding and base stacking forces
Backbone of sugar + phosphate

Information stored in 4 nitrogenous bases:
Adenine
Guanine
Thymine
Cytosine

Slide 14 L20

Question 16

How is DNA stored in cells?

Answer 16

Most DNA is present as large chromosomes (circular or linear)
Linear chromosomes are more complex due to end replication problems

Eukaryotes have linear genomic DNA chromosomes
Bacteria have single circular chromosome
(Some bacteria do have multiple chromosomes and/or linear chromosomes)

Question 17

What are plasmids?

Answer 17

Small rings of DNA that are extrachromosomal that add new properties to the cell

Bacterial chromosomes are several million based long while plasmids are sized in thousands of bases

Bacteria contain several hundred of copies of a single plasmid, bacteria divide very quickly

Putting a DNA sequence into a plasmid allows for rapid duplication of that sequence

Putting a gene into a plasmid May allow that gene product (protein) to be expressed at high levels

Question 18

What are the 5 plasmid types?

Answer 18

1. Conjugative plasmids- transmitted during conjugation, Carey variety of info
2. Resistance plasmids (R)- protect against environmental factors, MDR (multiple drug resistance) plasmids
3. Colicinogenic plasmids (Col)- codes for proteins that kill other microbes
4. Degradative plasmids- contain genes for novel catabolic enzymes
5. Virulence plasmids (vir)- increased the pathogenicity of a bacteria (ex: toxins)

Question 19

What is a cloning vector?

Answer 19

A vector is a plasmid thag has been streamlined and modified to make it amenable to carrying a payload of DNA

Origin of replication (make more)
Positive selection gene (only cells w/ vector live)
Insert differentiation gene (cells w/ insert look different)
Cloning sites (places to insert foreign DNA)

Question 20

What are the 5 steps to manipulate DNA?

Answer 20

1. Get plasmid pout of cells
2. Identify plasmids you want
3. Study/manipulate/disassemble DNA sequences
4. Reassemble DNA with new sequences
5. Put DNA back into cells

Question 21

What is a R/M system?

Answer 21

Host DNA is modified by methylation at a specific DNA sequence
Unmethylated DNA is restricted, or cut, at the same sequence if it is not methylated
Example S7L21

Question 22

What are the 3 restriction enzyme types?

Answer 22

Type I- R+M: a specific sequence is recognized by a multi subunit protein, a random sequence is cut hundreds of bases away
Type II- R: a specific sequence is recognized (4-8 bp palindrome) by a single protein and is cut within that sequence
Type III- R: a specific sequence is recognized by a multi-subunit protein, a random sequence is cut ~25bp away

Question 23

How do restriction enzymes cut?

Answer 23

They are molecular scissors
Cut within palindromic sequence
5’ staggered cuts
3’ staggered cuts
Blunt-ended cuts
Specific conditions needed: temp, [Mg], pH, [NaCl]

Question 24

What is a generic reaction?

Answer 24

4 components: DNA (vector or other), 10x enzyme buffer (salt, pH buffer, Mg+, BSA), water, enzyme
Buffer ensures optimal enzyme function
Water gets buffer to right concentration
Enzyme cuts the DNA

Assembled in order; enzyme always last

S10L21

Question 25

What is a double digest reaction?

Answer 25

Reactions can use 2 enzymes at once (if utilize compatible buffers)

Or reaction 1, stop, change buffer, reaction 2

Question 26

What is agarose gel electrophoresis?

Answer 26

Phosphate groups in DNA backbone give it a - charge
DNA + salt solution + electricity = DNA pulled to anode (+)
To slow/restrict passage of DNA it is placed in a gel matrix made of agarose (size purified agar)
S4L22

Question 27

How do you visualize agarose gel ladders?

Answer 27

2 ways
Ethidium bromide + DNA + UV light -> orange bands (dangerous carcinogen)
SyBr Green + DNA + blue light -> green bands (safer), is dangerous too but cant get into cells as easy
S5L22

Question 28

How do you size the bands from an agarose gel electrophoresis?

Answer 28

As distance is modified by light, T & V internal standards are needed
DNA ladder is known lengths to compare against
Bands must be linear (supercoiled circles travel faster than their apparent size)
Gel photographed next so a ruler

Question 29

How can you tell small and large pieces in agarose gel electrophoresis?

Answer 29

If pieces are too big or too small for the system we cant separate very well
Small pieces tumble - they twist and turn as they travel with no defined leading edge
Large fragments “reptate” - they use their leading edge to snake through the gel regardless of size
Lower concentration allows for large bands to be resolved while small bands run through
Higher concentration allows small bands to be resolved while large bands stack together

Question 30

How can you tell the direction a fragment is cloned in when doing restriction digest and plasmid testing?

Answer 30

slide 8 L22

Question 31

What is restriction mapping?

Answer 31

Used to identify/verify DNA pieces of interest
Uses a series of single and double digests to understand the locations of restriction sites relative to each other on a DNA fragment
Positions of cut sites relative to one another or known gene/sequence locations makes a map
S9L22

Question 32

What is the problem with big fragments in agarose gel electrophoresis?

Answer 32

Above ~20kb DNA fragments will not separate based on size due to reptation
Pulsed field gel electrophoresis alternates the direction of movement to counter reptation
Direction is switched every 10-60s
Fragments up to 1Mb can be resolved
S10L22

Question 33

How are DNA fingerprints created?

Answer 33

Cut bacterial chromosomal DNA w/ rare enzyme
Compare fragments to create strain-specific patterns
Unique fingerprint for that DNA
S11-12L22

Question 34

What is PulseNet?

Answer 34

Created to do surveillance and tracking of foodborne illnesses
Use PFGE to track strains and look for clusters or outbreaks (especially where non-localized clusters occur)