CHEM 1003 > Lecture 10 > Flashcards

Lecture 10 Flashcards

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1
Q

Electrophoresis separates DNA based off what

A

Charge

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2
Q

Agrose gel electrophoresis is useful for separating fragments of DNA that are at least ___ bp long and at least ____ bp different from other sized fragments

A

50; 50

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3
Q

How is agarose gel created

A

Agarose is a carb isolated from seaweed

Dissolved by boiling the powder in a buffer and then allowing it to polymerize

Poured into a tray with a comb that creates wells

Ends with a gel with a porous network where DNA can slip through the holes

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4
Q

An agarose gel has a positive and negative side - the gel is input in the ___ side and migrates toward the ____ side

A

Negative —-> positive

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5
Q

How is the density of the matrix (large or small holes) adjusted. How does this affect DNA travel

A

Via adjusting the % agarose - the lower the concentration, the larger the pores and the faster DNA will travel

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6
Q

Typically agarose gel concentrations range between:

A

0.75% to 2%

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7
Q

How is the desired density chosen for agarose gel?

A

Based on the size of the DNA fragment

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8
Q

2% gel is good for fragments of what size?

A

<400 bp

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9
Q

1% gel is good for fragments of what size?

A

400-1000bp

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10
Q

0.8% gel is good for fragments of what size?

A

> 1000bp (gDNA)

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11
Q

0.6% gel is good for fragments of what size?

A

It’s not! anything <0.7% is too flimsy or will melt with the electrical current

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12
Q

You need to prepare 100mL of a 1% m/v agarose gel for DNA electrophoresis. How much agarose powder in grams should you weigh out?

A

c= m/v
m = c/v

= 1g/100mL * 100mL
orrr
= 1g/dL * 10dL
= 10g

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13
Q

What colour is the cathode? What charge does it represent

A

black
negative

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14
Q

What colour is the anode? What charge does it represent

A

red
Positive

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15
Q

T/F

DNA runs anode to cathode

A

False

Cathode to anode

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16
Q

High quality DNA consists of short, intact strands of sharp-appearing nucleic acids on a gel

A

False (ish) - long strands

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17
Q

A blurry band/smear on an electrophoresis gel means the DNA is

A

Low quality; degraded

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18
Q

Where do larger fragments tend to land on the gel?

A

Closer to the top (migrate slower)

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19
Q

T/F

Fluorescent stains can be added to the gel both pre-gel and post-run

A

True - pre-gel more common with DNA though and post-run more common with protein staining

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20
Q

What are the two fluorescent stains we talk about

A

Ethidium bromide (EtBr)
Safe-red

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21
Q

Describe EtBr

A

Gold standard
Most common stain
Is an intercalator (inserts itself into spaces between bps)
Fluoresces in UV light
Potent mutagen
Carcinogenic

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22
Q

Describe Safe-red

A

Is an intercalator (inserts between bps)
Fluoresces in UV light
Non-carcinogenic

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23
Q

Why are buffers used in gel electrophoresis instead of water

A

Buffers optimize the pH and ion concentration
Allows the current to be evenly carried so DNA can run smoothly through the gel

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24
Q

What are the components in TBE? What does each do?

A

Tris - maintains pH
Boric acid: allows for stronger buffering capacity
EDTA - chelates Mg to inactivate nucleases

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25
What are the components in TAE? What does each do?
Tris - maintains pH Acetic acid - allows for more conductivity EDTA - chelates Mg to inactivate nucleases
26
What is one adv to TBE? One disadv?
More stable due to buffering capacity Slower DNA migration
27
What is one adv to TAE? One disadv?
Faster DNA migration Less stable (can overheat)
28
What are loading dyes?
Dyes that are mixed with glycerol or ficoll Added to DNA when pipetted in Keeps the DNA from floating out of the wells Allows us to visualize the experiment
29
What are some examples of loading dyes
Bromophenol blue Xylene cyanol Orange G (OG)
30
When do you know when to stop your gel electrophoresis current?
When the loading dye reaches the end of the gel
31
T/F Never blow out the pipette when loading DNA samples into electrophoresis gel
True
32
T/F The voltage of the gel determines how far the DNA runs
False - the size of the fragment does
33
T/F DNA has different charge depending on size
False - it has an equivalent charge no matter the size
34
What is the best way to monitor if your electrophoresis is running?
Look for bubbles - if present - it's on
35
What is a molecular ladder
A set of DNA fragments of known molecular size Like a QC - required to report out results Used as a standard to determine the sizes of unknown fragments being tested
36
What would be used for a positive control on a gel electrophoresis
A DNA fragment of known size
37
If you have a patient sample whose band pattern matches one of your positive controls - what does this mean
The patient is positive for whatever that control is
38
T/F Gel electrophoresis may have multiple positive controls, if looking for multiple targets
True
39
T/F Gel electrophoresis positive controls may have positive controls with multiple bands, if looking for multiple targets
True
40
T/F The molecular ladder is determined in lab before each experiment
False - it is given by the manufacturer
41
What 4 gel electrophoresis methods do we learn about regarding protein separation
SDS page + Western blot Native gel electrophoresis Capillary electrophoresis Isoelectric focusing
42
Describe SDS Page + Western blot
Denatures proteins into a straight, linear piece Can then be used for serum protein electrophoresis (SPEP) where patterns of bands are identified to assess abnormalities
43
Native gel electrophoresis involves protein moving through the gel based on ____
shape
44
Which of the four gel electrophoresis for proteins is used most commonly in hgih-throughput chem labs
Capillary electrophoresis
45
Describe how polyacrylamide gel differs from agarose gel
Polyacrylamide gel is used for SDS-PAGE and Native Page Is vertically placed, and wells are loaded on the top and migrate down (instead of horizontal) Can be used for DNA or protein, but more often protein Staining is done post-run (rather than during or before run)
46
What is the most commonly used denaturant in SDS-PAGE
SDS lol
47
SDS-PAGE stands for...
Sodium Dodecyl Sulfate-PolyAcrylamide Gel Electrophoresis
48
What does SDS do?
Gives a neg charge to each protein in proportion to its mass Helps with protein unfolding Works with other chemicals to denature protein into linear structure
49
SDS-PAGE separates proteins based on what
Weight (molecular mass)
50
Incomplete denaturation of proteins will lead the gel bands to look like what?
Blurry
51
What is one limitation to SDS-PAGE
Cannot differentiate between proteins of same molecular mass
52
Describe Native PAGE
Method of protein electrophoresis that allows examination of proteins in their natural state (not denatured) Separated based on shape Can separate proteins of identical molecular weirds bc shapes are different
53
What happens if you apply heat to Native PAGE?
Proteins will denature and she will not work
54
T/F protein gel electrophoresis stains can stain specific targets
False - not sensitive, will stain all proteins
55
What are some protein gel electrophoresis stains
Coomassie Brilliant Blue & Ponceau S Silver Stain
56
What protein gel electrophoresis stain is sensitive, but disallows for downstream applications as it destroys DNA
Silver stain
57
T/F Western blotting is used to detect specific proteins
true
58
V briefly describe the process of western blotting
Gel is placed on a membrane of nitrocellulose paper Electrotransfer gel onto membrane Visualize via probe, staining, etc (requires further processing)
59
To visualize western blots we use what?
Secondary antibodies, conjugated with an enzyme or label
60
What are the 4 ways you can visualize a western blot. Briefly describe each
Colorimetric - signal is a coloured precipitate produce Chemiluminescence - reaction produces a light as a byproduct of the substrate catalysis Fluorescence - antibody is labeled with a fluorophore Radioactive - antibody is radioisotope labelled, uses X-ray to produce an image
61
What is the purpose of blocking
Prevents non-specific binding of antibodies to the gel itself in western blotting
62
Why must we wash between primary and secondary antibody additions in western blotting?
To prevent non-specific fluorescence
63
Southern blots detect what?
Specific sequences of DNA
64
Southern blots use what kind of enzymes?
Restriction enzymes - creates DNA fragments and separates into ssDNA
65
Southern blotting separates DNA by ____
size
66
Northern blots detect what?
RNA fragments
67
Capillary electrophoresis separates charged molecules based on what?
Their size-to-charge ratio
68
____ charge, ____ size means molecules will move faster through the capillary tube in capillary electrophoresis
High; small
69
___ charge, ____ size means molecules will mvoe slower through the capillary tube in capillary electrophoresis
Low; large
70
When are restriction enzymes used in the lab
Before DNA is run through gel electrophoresis - cuts it up into smaller fragments RFLP (detects genetic mutations, HLA typing, disease carrier testing) Subtyping microbial bacterial strains Oncology
71
T/F Different restriction enzymes are always made to cut different, specific DNA sequences
True
72
What is recombinant DNA
DNA fragments from two unrelated organisms that have been cut by restriction enzymes and pieces together
73
Clinically, what is recombinant DNA used in?
Insulin Factor replacement for hemophilia Human growth hormone Vaccines Gene therapy
74
Sequencing allows us to....
Determine the exact order of nucleotifes in DNA or RNA Identify mutations
75
What are two sequencing methods
Sanger sequencing Next generation sequencing
76
Which type of sequencing is better for large high throughput analysis
NGS
77
Sanger Sequencing has all the same reagents as PCR except with the addition of ____
ddNTPs
78
Describe ddNTPs
Fluorescently labelled nucleotides Lack OH group required for more nucleotides to bind
79
T/F All ddNTPs have the same fluorescence
False - each produce a diff colour (ex: ddAPT is red, ddCTP is blue)
80
Outline the steps involved in sanger sequencing
PCR is run with ddNTPs in addition As polymerase adds dNTPs during PCR elongation (after multiple copies have been done), it will randomly add a ddNTP and terminate the sequence You will have many copies of incomplete fragments of varying sizes Size separation via gel capillary electrophoresis Gel analysis and determination of DNA sequence via a laser
81
T/F NGS reads thousands of DNA fragments all at once
True
82
Outline the 4 steps of NGS
1. Library preparation -> extract DNA and attach adaptors to "barcode" the target sequence until whole sequence is chopped and barcodded 2. Amplification -> Lots of copies are made 3. Sequence the library -> Find the first barcode, read... next barcode, read.... all in order. Combine all the fragments together at the end in the correct order 4. Data analysis -> Analyze the whole combined sequence and identify diseases and/or mutations
83
Describe pyrosequencing
Real-time sequencing by synthesis method As dNTPs are added, DNA pol releases a pyrophosphate This pyrophosphate goes through additional reactions and produces a light that is measured and used to determine sequence
84
On a pyrogram, the y-axis represents...
light intensity
85
On a pyrogram, the x-axis represents
dNTPs it tried to attach
86
T/F If DNA pol added a nucleotide and a peak appeared on the pyrogram, what does this mean?
That nucleotide is present in the sequence at spot
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