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BMSC 240 > Final Exam (cumulative) > Flashcards

Final Exam (cumulative) Flashcards

1
Q

What is the “Bottom-up” method?

A

Look at a system starting at its molecular level, and ending at the ecosystem as a whole

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2
Q

What is the “Top-down” method?

A

Look at a system starting at the ecosystem as a whole, and ending at its molecular level

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3
Q

What is sonication?

A

Using sonic/sound/vibration to rip cells apart; vibration also makes heat

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4
Q

What is the French Press?

A

Using pressure to “pop” the cells open

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5
Q

What is Osmotic Shock?

A

Low salt solution, this is not effective for regular cells walls, but is used for RBCs

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6
Q

What is Digestion?

A

Using enzyme digests

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7
Q

What are Detergents?

A

Break up fats and oil, break up the phospholipid bilayer

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8
Q

What is Homogenization?

A

Using brute force, like a blender/grinder for bigger chunks of solids

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9
Q

Proteins removed from a cell degrade by the non-_______ conditions

A

physiological

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10
Q

In extract stabilization, care should be taken to avoid 4 things: ?

A
  1. pH changes
  2. Heat denaturation
  3. Oxidation
  4. Proteolysis
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11
Q

What would solve the issue of pH changes in extract stabilization?

A

Extract/keep in buffered solutions

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12
Q

What would solve the issue of pH changes in heat denaturation?

A

Keep sample cold, heat only as needed

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13
Q

What would solve the issue of pH changes in oxidation?

A

Reducing agents can be added to prevent spontaneous crosslinking

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14
Q

What would solve the issue of pH changes in proteolysis?

A

Lower temperature, or add protease inhibitors

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15
Q

What are proteases?

A

Enzymes that break down proteins, proteolysis

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16
Q

To separate the protein/enzyme you want from everything else you have to be able to track it using an ____

A

assay

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17
Q

Assays should be
– _____
– _____
– ____
– ______

A

Sensitive, Specific, Rapid, Quantitative

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18
Q

What is Activity in terms of Purification Tracking?

A

how many U of enzyme per mL

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19
Q

What is Specific Activity in terms of Purification Tracking?

A

how many U per mg protein

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20
Q

What is Yield in terms of Purification Tracking?

A

(total Activity after purification) / (total Activity before
purification)

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21
Q

What is Purity in terms of Purification Tracking?

A

(S.A. after) / (S.A. before)

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22
Q

What is a method based in Solubility for Purification by physical properties?

A

Salting out

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23
Q

What are 3 methods based in Charge for Purification by physical properties?

A

Ion exchange chromatography
Electrophoresis
Isoelectric focusing

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24
Q

What is a method based in Polarity for Purification by physical properties?

A

Reverse phase (hydrophobic) chromatography

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25
Q

What are 3 methods based in Size for Purification by physical properties?

A

Dialysis
Size-exclusion chromatography
Gel electrophoresis

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26
Q

What is a method based in Binding Specificity for Purification by physical properties?

A

Affinity chromatography

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27
Q

What is a method of initial purification?

A

Centrifugation

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28
Q

If the protein is soluble it can be partially purified by removing larger/denser contaminants by differential _______

A

centrifugation

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29
Q

In centrifugation, the faster the spin, the _____ the contaminants can be removed

A

smaller

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30
Q

During differential precipitation, the supernatant (liquid at the top of the centrifuge tube) can be further treated to make some of the proteins in the mixture ____ soluble, by altering the _______, _____ or ___

A

less, temperature, salinity, pH

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31
Q

As [salt] _____, the + & - ions will compete with the hydrophilic surface amino acids for water the protein molecules with insufficient hydration will aggregate & lose _____

A

increases, solubility

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32
Q

In dialysis, excess salt must be ______ as it reduces solubility and can impede subsequent isolation steps

A

removed

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33
Q

Dialysis tubing has pores with a specific molecular
weight cut-off that allow _____ molecules (salt) to pass.

A

smaller

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34
Q

Dialysis is great for separating proteins from salts, but not for separating proteins themselves based on ____

A

size

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35
Q

________ is a technique used to separate mixtures based on physical properties such as size or charge

A

Chromatography

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36
Q

What are the two phases in chromatography?

A

Stationary and mobile

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37
Q

What is the stationary phase in chromatography?

A

A substance that the compounds to be separated pass by or interact with

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38
Q

What is the mobile phase in chromatography?

A

The carrier for the compounds to be separated

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39
Q

In chromatography, a mixed sample needs to have _______ affinity, some sample may have higher affinity for the _____ phase, some sample may have higher affinity for the _______ phase

A

variable, mobile, stationary

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40
Q

With different affinities in chromatography come different rates of migration = ?

A

separation of the molecules

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41
Q

What are the 5 basic steps in column chromatography?

A
  1. Pouring
  2. Packing
  3. Loading
  4. Running/eluting
  5. Collecting
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42
Q

In what kind of chromatography do the porous beads make up the stationary phase?

A

Size exclusion

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43
Q

In size exclusion chromatography, there are several important volume concepts:
– Vt:
– Vo:
– Vi:
– Ve:

A

The volume of the column
The volume outside the beads
The volume inside the beads
The volume at which a sample is eluted

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44
Q

In size exclusion chromatography, _____ proteins can not enter the beads so they will migrate around them so they travel in only the ___

A

large, Vo

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45
Q

In size exclusion chromatography, _____ sized proteins enter beads some times which increases the volume available and move more slowly so they travel in ____ and some ___

A

moderate, Vo, Vi

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46
Q

In size exclusion chromatography, small proteins do not interact with the beads and move the slowest so there is no Vo or Vi, just ____

A

Vt

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47
Q

The _______ _______ is the fraction of the pores within the beads available to the sample

A

partition coefficient

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48
Q

Kav = __ = always excluded by beads
Kav = __ = beads fully accessible

A

0, 1

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49
Q

What is the formula for Kav?

A

Kav = (Ve – Vo)/(Vt - Vo)

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50
Q

Partition coefficient can be used to estimate ?

A

molecular weight

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51
Q

RMW = relative molecular weight – mainly affected by protein _____, but also the _____ of the protein of interest may be different from the size standards causing it to run faster/slower than the actual molecular weight

A

length, shape

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52
Q

If Kav is >0.8 or <0.2 you will get ____ separation of proteins

A

poor

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53
Q

Samples become diluted as:
______ from layering sample over gel
______ during travel
______ differences along the glass tube wall

A

Turbulence, Diffusion, Friction

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54
Q

A column of positively or negatively charged beads forms the stationary phase.
_____ exchange (column is +, targets are -)
_____ exchange (column is -, targets are +)

A

Anion, Cation

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55
Q

In ion exchange chromatography, proteins in solution get carried down by gravity and will get slowed by the beads proportionally based on _____

A

charge

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56
Q

In ion exchange chromatography, the gradient elution can be done using a simple __ beaker, tube and stirbar setup. As the elution will occur at the top of the column first and work downwards, this is a ______ column

A

2, focusing

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57
Q

In ion exchange, there is such thing as a spin column: ion exchange columns give a fast/clean method to extract DNA from ?. Mini column is loaded, spun, washed, spun, eluted, spun

A

cleared bacterial lysate

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58
Q

In _____ chromatography, there is a specific trap for protein of interest attached to stationary phase
– enzyme ‒> substrate
– receptor ‒> hormone
– antigen ‒> antibody
– (His)6 protein –> Ni2+
But elution can be problematic, yet high purity can be achieved

A

affinity

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59
Q

Order the 3 kinds of chromatography from most to least specific: ?

A
  1. Affinity chromatography
  2. Ion exchange chromatography
  3. Size exclusion chromatography
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60
Q

_______: migration of ions in an electric field

A

Electrophoresis

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61
Q

In electrophoresis, velocity is directly proportional
to ____ and inversely proportional to ____ and ___

A

charge, size, shape

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62
Q

In _____ gel electrophoresis, proteins have inherent charge differences but may also be coated with charged particles. Differences in size and charge can make determining why a protein moved fast/slow difficult

A

protein

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63
Q

A gel is used to ?, ?, and _____ ______

A

slow down movement, prevent diffusion of the proteins, create separation

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64
Q

What does SDS-PAGE stand for?

A

Sodium dodecyl sulfate polyacrylamide gel
electrophoresis

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65
Q

SDS = a _____ that will denature proteins and give them a negative charge

A

detergent

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66
Q

~____ g SDS / ___ g protein

A

1.4, 1.0

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67
Q

Monomers are ______, _______ and ______ , but once polymerized it’s safe

A

neurotoxic, carcinogenic teratogenic

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68
Q

Acrylamide (acrylic amide) = a plastic ______

A

monomer

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69
Q

Acrylamide: forms ____

A

chains

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70
Q

Bis-acrylamide: branch _____/_______

A

chains, crosslinking

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71
Q

Acrylamide + catalyst + a suitable buffered salt solution = _____

A

gel

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72
Q

Around a __:__ mix of acrylamide to bisacrylamide

A

30:1

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73
Q

What are the 5 basic steps in the SDS-PAGE procedure?

A
  1. Casting the gel
  2. Forming the wells with a comb
  3. Preparing the gel
  4. Running the gel
  5. Visualizing the gel
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74
Q

Protein samples are boiled for __ minutes in __x loading buffer

A

5, 5

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75
Q

In the SDS-PAGE procedure, there is :
__% SDS (denatures protein)
__% glycerol (adds density & viscosity)
___M Tris-HCl pH 6.8 (buffer)
___% bromophenol blue (tracking/loading dye)
__mM β-mercaptoethanol (break disulfide bonds

A

10, 20, 0.2, 0.05, 10

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76
Q

In the SDS-PAGE procedure, there are 3 different dyes that can be used, what are they?

A
  1. Coomassie blue (fastest and least sensitive)
  2. Silver nitrate (most complicated, most sensitive)
  3. Fluorescent dye staining (sensitive, requires special equipment)
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77
Q

Describe the protocol of silver staining: ?

A

Requires more than half a dozen unique solutions to turn a gel into a piece of black & white photographic film

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78
Q

In protein sizing, ____ proteins are less mobile, __ proteins are more mobile, and the ____ of known sizes is used to figure out the sizes of other bands

A

bigger, smaller, ladder

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79
Q

Sometimes linearity is not observed on a standard curve: if at large sizes, need ____ % gel, but if at small sizes, need ____ % gel, and if mid-range, could be due to intrinsic charge or protein folding

A

lower, higher

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80
Q

The height of a well can produce a vertically diffuse band, there are 4 solutions to this: ?

A
  1. Use less volume per sample
  2. Concentrate the sample
  3. Run a vertically longer gel
  4. Run a stacking gel
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81
Q

The height of a well can produce a vertically diffuse band. one solution is to use less volume loaded per sample, but what is the downside to this?

A

The bands will be faint

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82
Q

The height of a well can produce a vertically diffuse band. one solution is to concentrate the sample, but what is the downside to this?

A

Much more time consuming

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83
Q

The height of a well can produce a vertically diffuse band. one solution is to run a vertically longer gel, but what is the downside to this?

A

More time consuming, requires a non-standard apparatus

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84
Q

The height of a well can produce a vertically diffuse band. one solution is to run a stacking gel, but what is the downside to this?

A

Required a special 2-layer gel

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85
Q

2 gels + 2 buffers + 3 pH’s = _____ solution

A

elegant

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86
Q

Heating and running samples in a gel made with ~6 M urea allows for proteins to denature but retain their _____ _____

A

native charge

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87
Q

In _____ gels, mobility of proteins depends on size, charge and conformation (shape), and can be used to keep protein complexes intact

A

native (Non-denaturing)

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88
Q

In native gels, charge depends on the ___ of the buffer used

A

pH

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89
Q

Sickle-cell anemia has a single amino acid change in hemoglobin A and the charge differences show up nicely on ____ gels without breaking the hemoglobin tetramer

A

native

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90
Q

____ hemoglobin has a higher affinity for oxygen than _____

A

fetal, adult

91
Q

In ____ _______ electrophoresis, proteins are separated based on 2 factors, in 2 dimensions, to allow us to visualize the contributions of size on one, charge on the other

A

2-D protein

92
Q

In 2-D protein electrophoresis, what is Dimension 1?

A

Isoelectric Focusing Gels

93
Q

_____ are molecules that can buffer at their pI

A

Ampholytes

94
Q

______ _____ gels requires a non-diffusing pH gradient, and contains a series of ampholytes are embedded into the gel which allows them to maintain a pH gradient without moving during electrophoresis

A

Isoelectric Focusing

95
Q

In ______ _____ gels, proteins are electrophoresed
through the ampholyte-containing gel until they hit their pI, where they become uncharged and stop

A

Isoelectric focusing

96
Q

In 2-D protein electrophoresis, what is Dimension 2?

A

SDS-PAGE

97
Q

In _____ gels, isoelectric Focusing Strips are treated to denature proteins and coat with SDS. Now-uniformly-negative SDS+proteins are next resolved by size

A

SDS-PAGE

98
Q

What kind of gel can rapidly separate thousands of proteins in a mixture to individual spots?

A

2D

99
Q

What kind of gel can identify samples from different sources can be compared to determine what proteins are different?

A

2D

100
Q

What kind of gel can have spots that can be excised and identified if necessary?

A

2D

101
Q

_____ as a group they recognize antigens, but specifically they each bind a single epitope

A

Antibodies

102
Q

Mammals make 5 varieties of antibodies that vary by their tail (immunoglobulins): ?

A
  1. IgG
  2. IgM
  3. IgA
  4. IgD
  5. IgE
103
Q

What is an IgG?

A

Gamma globulin

104
Q

___ are monomers, bivalent, and are about 80% of serum antibodies.

A

IgG

105
Q

___ recruit the immune system, are in the blood, lymph, and intestine, and can cross the placenta

A

IgG

106
Q

____ enhance phagocytosis; neutralize toxins and viruses; protects fetus and newborn, and has a half-life of 23 days

A

IgG

107
Q

Immature B cells have several __, __ and __ DNA sequences – Like having multiple items of pants, shirts & shoes to make up a diversity of outfits

A

V, D, J

108
Q

Random _____ in the light and heavy chains make unique antibodies

A

deletions

109
Q

In B-Cell clonal selection, there are 2 tests:
Test #1 – ?
If yes, kill cell
Test #2 – ?

A

does the B-cell make host binding antibodies?
does the B-cell make useful antigen binding antibodies?

110
Q

Naïve B-cells will be activated upon antigen exposure to form ______ ___ or _____ _____

A

plasma cells, memory B-cells

111
Q

Antibody ____ rises and falls

A

titer

112
Q

______ boosts the amount of antibodies produced. Memory B-cells will retain specific Ab potential for a ____ (or less)

A

Reinfection, lifetime

113
Q

What are the 5 protective mechanisms of binding antibodies to antigens in “in vivo”?

A
  1. Neutralization
  2. Agglutination
    3 Opsonization
  3. Activation of complement
  4. Antibody-dependent cell-mediated cytotoxicity
114
Q

What is neutralization?

A

Blocks adhesion of bacteria and viruses to mucosa; blocks attachment of toxin

115
Q

What is agglutination?

A

Reduced number of infections units to be dealt with

116
Q

What is opsonization?

A

Coating antigen with antibody enhances phagocytosis

117
Q

What is Activation of Complement?

A

Causes inflammation and cell lysis

118
Q

What is Antibody-Dependent Cell-Mediated Cytotoxicity?

A

Antibodies attached to target cell cause destruction by eosinophils and NK cells

119
Q

Secondary antibodies can be produced in a different organism using the tail (Fc) from _____ antibodies

A

primary

120
Q

Tails are shared similarity by _____, humans are the same, but not compared to hamster, etc

A

species

121
Q

______ antibodies: a natural immune reaction, several B-cells each make a different antibody that recognizes the antigen
______ are single B-cells made immortal so a single epitope is recognized and available for a long time

A

Polyclonal, Monoclonal

122
Q

In ______ production, cells fused with cancerous B-cells to make them immortal, metabolic & immunological selection for fused cells. The result is called a hybridoma

A

monoclonal

123
Q

_____ Blotting: a method to look for a protein of interest from a gel using antibodies against the protein

A

Western

124
Q

Identify what method this is:
A polyacrylamide protein gel is first run, the gel is transferred to a membrane with a high affinity for protein (nylon or nitrocellulose), which is treated to bind the antibody of interest and visualized

A

Western Blotting

125
Q

The membrane in Western Blotting has a high affinity for protein, so all empty spots must be filled before antibodies can be added. Once blocked the 1° antibody is added to the blot and allowed to bind. A 2° antibody may then used to ?

A

detect the presence of the 1° antibody

126
Q

What are the 4 types of antibody labels?

A
  1. Isotope labelling
  2. Enzyme labelling
  3. Fluorescent labelling
  4. Heavy element labelling
127
Q

What kind of antibody labelling is described?

125I or 35S are attached to the protein, as the isotope decays the electrons expose X-ray film layered over the blot

A

Isotope Labelling

128
Q

What kind of antibody labelling is described?

Common enzymes like horseradish peroxidase (HRP) or alkaline phosphotase (AP). The enzyme catalyzes a reaction to give a coloured substance or chemiluminescence

A

Enzyme Labelling

129
Q

What kind of antibody labelling is described?

A dye is attached that absorbs one color of light and gives off another

A

Fluorescent labelling

130
Q

What kind of antibody labelling is described?

Metals or rare earth elements that are detectable by electron microscopy

A

Heavy element labelling

131
Q

The following are applications for what method?
Detection (and quantification) of a protein from a sample, expression profiles of a protein in tissue samples, verification of a transgenic organism, detect infections

A

Western Blot

132
Q

What does ELISA stand for?

A

Enzyme-linked-immunosorbent assay

133
Q

ELISA steps similar to western blots, but with no ______ _____

A

protein separation

134
Q

What is the difference between an Antigen and an Epitope?

A

An epitope is that part of the antigen to which antibodies bind. While the antigen evokes the antibody response in the host, the antibody doesn’t bind to the entire protein, but only to that segment called the epitope

135
Q

? aka lateral flow tests have been developed to detect pathogens, blood, drugs, genetically modified foods and more. In recent years, the “enzyme” of ELISA has been substituted with gold nanoparticles for these tests

A

Rapid strip tests

136
Q

________ test are used to test cell specimens, but especially bacterial and blood cells

A

Agglutination

137
Q

In an ________ test, an emulsion of cells is added to an antibody mixture and rocked back and forth

A

agglutination

138
Q

Agglutination tests can be done in 2 ways: ?

A

Unknown cells with specific antibody
Known cells with unknown serum

139
Q

The precipitin Reaction and the ouchterlony assay are both under the category of what kind of assay?

A

Diffusion

140
Q

_______ assays are useful when samples are too small to agglutinate visibly

A

Diffusion

141
Q

What kind of method is being described?

A ring of wells is cut into an agar plate around a central well, with the antibodies/serum in the center. Ab & Ag diffuse through the agar and will form a visible precipitate between the wells if they are compatible. The continuity of the line between samples can indicate shared antigens

A

Diffusion assay

142
Q

What kind of method is being described?

Cultured cells or tissue sections are placed on slides, they are then fixed using paraformaldehyde or methanol/acetone & rinsed. Cells are permeabilized using dilute detergent and are blocked to reduce nonspecific binding. Primary antibody is added / washed, then the secondary fluorescent antibody is added / washed

A

Immunofluorescent Staining + Fluorescent Microscopy

143
Q

What are the 3 kinds of fluorescent microscopy?

A
  1. Standard
  2. Confocal
  3. Two-photon
144
Q

What is Standard fluorescent microscopy?

A

Whole image is illuminated and out-of-focus light makes the in-focus image fuzzy

145
Q

What is Confocal fluorescent microscopy?

A

Whole image is illuminated and out-of-focus light is blocked

146
Q

What is Two-photon fluorescent microscopy?

A

Less energetic light is used (infrared) and 2 photons must hit the dye simultaneously to excite it

147
Q

What are the pros and cons of Standard fluorescent microscopy?

A

Pro: cheap
Con: fuzziness and photodamage

148
Q

What are the pros and cons of Confocal fluorescent microscopy?

A

Pro: very good resolution
Con: photodamage

149
Q

What are the pros and cons of Two-photon fluorescent microscopy?

A

Pro: lower photodamage, better tissue penetration
Con: costliest, inferior resolution to confocal

150
Q

Which of the 4 nitrogenous bases are purines?

A

Adenine and guanine

151
Q

Which of the 4 nitrogenous bases are pyrimidines?

A

Thymine and Cytosine

152
Q

The duplex of DNA is held together by _____ bonding and ____ ______ forces

A

hydrogen, base stacking

153
Q

What are the 5 different plasmid types?

A
  1. Conjugative (F)
  2. Resistance (R)
  3. Colicinogenic (Col)
  4. Degradative
  5. Virulence (vir)
154
Q

What are Conjugative plamids (F)?

A

Transmitted during conjugation, carry a variety of information

155
Q

What are Resistance plamids (R)?

A

Protect against environmental factors, MDR (multiple drug resistance) plasmids

156
Q

What are Colicinogenic plasmids (Col)?

A

Codes for proteins that kill other microbes

157
Q

What are Degradative plamids?

A

Contain genes for novel catabolic enzymes

158
Q

What are Virulence plamids (vir)?

A

Increases the pathogenicity of a bacteria

159
Q

LF+PA = a ____ toxin which kills human cells

A

lethal

160
Q

EF+PA = _____ toxin which causes cells to leak
cytoplasm

A

edema

161
Q

Cap(A-E) = ____ formation to avoid immune system detection

A

capsule

162
Q

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

A

vector

163
Q

Here are the typical components of a cloning vector:
– _____ of replication
– _______ selection gene
– Insert _______ gene
– _____ sites (places to insert foreign DNA)

A

Origin, Positive, differentiation, Cloning

164
Q

Order these statements in accordance to the Basic Cycle of DNA (sub)cloning:
1. Identify the plasmids
2. Put DNA back into cells
3. Get plasmids out of cells
4. Study/manipulate/disassemble DNA sequences
5. Reassemble DNA with new sequences

A

3, 1, 4, 5, 2

165
Q

What is the R/M system?

A

Restriction/Modification system of bacteria
Host DNA is modified by methylation at a specific DNA sequence, and unmethylated DNA is restricted, or cut, at the same sequence if it is

166
Q

What are the 3 types of Restriction Enzymes?

A
  1. Type I
  2. Type II
  3. Type III
167
Q

Type ___ restriction enzyme: R+M - a specific sequence is recognized by a multi-subunit protein, a random sequence is cut hundreds of bases away

A

I

168
Q

Type ___ restriction enzyme: R - a specific sequence is recognized (4-8 bp palindrome) by a single protein and is cut within that sequence

A

II

169
Q

Type ___ restriction enzyme: R - a specific sequence is recognized by a multi-subunit protein, a random sequence is cut ~25 bp away

A

III

170
Q

What is the difference between sticky and blunt ends of restriction enzymes?

A

Sticky ends are cuts of DNA that have DNA fragments on either side of the cut made by the restriction enzyme.
Blunt ends cut DNA symmetrically and there is no overhang on either side of the cut.

171
Q

_____ digest: reactions can use 2 enzymes at once

A

Double

172
Q

If a double digest is incompatible, digests can be
done sequentially: ?

A

reaction 1 → stop (heat denature enzyme) →
change buffer → reaction 2

173
Q

Fill in the blanks:

We have a plasmid that is 4kb & we know the locations of these restriction sites relative to this “map” & we’re interested in isolating. The ___ is our start, the numbers listed are not ______ from one position to the next, they are absolute positions. The different fragments are generated with EcoRI, KpnI, or both, and these fragments will also have _____ ends (potential overhangs created by the enzyme)

A

top, distances, different

174
Q

DNA + salt solution + electricity = DNA pulled to ?

A

Cathode (+)

175
Q

In an agarose gel, the gel immersed in buffer, what are the 3 most commonly used?

A

TAE, TBE, SB

176
Q

_______ Agent: will be flat and insert itself into the laddered runs of DNA in between the base pairs

A

Intercalating

177
Q

When we want to visualize the DNA:
_______ ______ + DNA + UV light → orange bands
____ _____ + DNA + blue light → green bands

A

Ethidium bromide, SyBr Green

178
Q

When sizing bands in an agarose gel, the bands must be ____, as supercoiled circles travel ____ than their true size, and relaxed circles travel ____ than their true size

A

linear, faster, slower

179
Q

In an agarose gel, small pieces “_____” – they twist and turn as they travel with no defined leading edge – if too small, the gel doesn’t slow similarly sized small pieces. Large fragments “____” – once they start to migrate they use their leading edge to snake through the gel regardless of size

A

tumble, reptate

180
Q

In an agarose gel, a ____ concentration allows for large bands to be resolved while small bands run through; a _____ concentration allows small bands to be resolved while large bands stack together

A

lower, higher

181
Q

What is Star Activity?

A

Under non-standard reaction conditions, some restriction enzymes are capable of cleaving sequences which are similar, but not identical, to their defined recognition sequence.

182
Q

How do we solve the issue of having fragments that are too large?

A

Pulsed field gel electrophoresis alternates the direction of movement to counter reptation. The direction is switched every 10-60s and fragments up to 1Mb can be resolved, but special extraction needed to avoid shearing

183
Q

We can cut bacterial chromosomal DNA w/ rare enzyme and compare fragments to create ?

A

strain → specific patterns, a unique “fingerprint” for that DNA

184
Q

____ is an enzyme which connects the phosphodiester backbone of DNA between a 3’-OH and 5’-P

A

Ligase

185
Q

Fragments to be assembled (vector and insert) are mixed with _____ under the right enzymatic conditions and within minutes to hours the DNA ends are linked together

A

ligase

186
Q

Assembled plasmids are mixed with ? that is chemically treated to become permeable. They then receive a “heat shock” at ___°C for __-__s causes some cells to take up plasmid from surrounding media, and the cells are allowed to recover before plating

A

E. coli, 42, 30-90

187
Q

A colony on a plate = one _______ event

A

transformation

188
Q

To analyze the plasmid more bacteria are
needed than one colony can provide, so single colonies are placed in LB Broth w/ antibiotic which
provides continual _____ to maintain plasmid

A

pressure

189
Q

What are the 4 most common antibiotics?

A

Ampicillin, Kanamycin, Tetracycline, Chloramphenicol

190
Q

What are the 5 steps in a miniprep?

A
  1. Extracting plasmid
  2. Resuspended
  3. Lysed by NaOH, SDS, boiling
  4. Precipitate debris – neutralize NaOH
  5. Spin out debris
191
Q

Similar to protein extractions: use _____, _______, ____ to break apart cells

A

chemicals, temperature, force

192
Q

Solvent extraction needs ______/______ to remove contaminants; and differential precipitation using _____ and _____

A

phenol/chloroform, salts alcohol

193
Q

? : is a method used to allow studying DNA in an un-cloned state by selective copying and amplification

A

PCR

194
Q

The basic components if the PCR mechanism include 5 things: ?

A
  1. Template DNA
  2. Oligonucleotide primers (~20 nt long)
  3. Deoxyribonucleotide triphosphates (dNTP)
  4. Heat-stable DNA polymerase (Taq polymerase)
  5. Suitable buffer solution
195
Q

What is the formula for PCR, and what does each components signify?

A

of copies = (2^n– 2n)x
Where n = # of cycles
x = # of copies to start

196
Q

What are the 3 basic steps in the first step of PCR?

A

Melt/denaturation: 94-95°C for 30-120s
Anneal: 45-65°C for 30-120s
Extend: 72°C

197
Q

In the PCR reaction mixture, there is a large range of template DNA, ranging from 10pg - 1µg, why is this?

A

Because a plasmid can equal one copy of sequence, a genome can equal one copy of the sequence

198
Q

What does PCR stand for?

A

Polymerase Chain Reaction

199
Q

Amplification will ______ as template binds template instead of primer at later cycles of PCR

A

plateau

200
Q

_____ polymerase: first discovered (cheapest), 5’ to 3’ polymerase without 3’ to 5’ exonuclease

A

Taq

201
Q

____ polymerase: somewhat costlier, but more processive (longer pieces can be amplified), fixes errors 25X more often (3’-5’ exonuclease), no 3’ A overhangs

A

Pfu

202
Q

__-__ bp is optimal for a primer

A

18, 24

203
Q

_____: avoid repeated sequences, ~50% GC, GC 5’ end clamp, pairs +/- 2°C, avoid hairpins and primer dimers

A

Primers

204
Q

______ are essential with such a sensitive method to amplify DNA, _____ ones are the easiest to do

A

Controls, negative

205
Q

What are the 3 kinds of thermocyclers/PCR machines?

A

Basic, gradient, real-time

206
Q

What is a Basic thermocycler?

A

Every tube cycles together

207
Q

What is a Gradient thermocycler?

A

Allows optimization of annealing temps

208
Q

What is a Real-Time thermocycler?

A

Allows quantification of each cycle

209
Q

____ is the temperature that a double stranded sequence of the primer would fully melt into single stranded molecules

A

Tm

210
Q

Annealing temperature of a PCR cycle is generally initially tested as 5°C below ___

A

Tm

211
Q

What is the original formula for calculating Tm?

A

Tm = 2°C x (A+T) + 4°C x (G+C)

212
Q

What is the problem with the original formula for calculating Tm?

A

Does not have an upper limit, so it is only valid for about 20bp

213
Q

What is the benefit for the more complicated formula for Tm?

A

Takes into account the salt concentration and order of the bases

214
Q

PCR is sensitive enough that you can amplify false positives because of contaminating DNA from: ?

A

You
Your pipette
The water
Other random errors

215
Q

Primers are designed to span genome regions that contain ______ sequences, which are chosen because different repeat numbers are found in a population. While one region may have few possibilities, observing many regions at a time can create a _____ combination

A

repeated, unique

216
Q

What does CODIS stand for?

A

Combined DNA index system

217
Q

CODIS loci has ___ primer sets, that were chosen from a much larger set with these goals in mind:
- The locations will independently assort during _____
- The locations are not linked (near) genes for visible human _______
- That the sizes are stable enough that children _____ repeat lengths from their parents

A

13, meiosis, phenotypes, inherit

218
Q

CODIS Markers can identify ______ individuals

A

Unique

219
Q

Blood can be used for exclusion, we can determine it (may/may not?) be you

A

may not

220
Q

Primers can be designed to give a product that is cut/not cut with a restriction enzyme to reflect presence of a _____ gene. PCR can _____ a disease region to be used for detailed analysis – full sequencing of the region

A

disease, amplify

221
Q

A primer can be developed with a ____ nucleotide mismatch, or a small insertion/deletion off a plasmid. The amplified product is then isolated / transformed / studied / mutagenized even further

A

single,

222
Q

_____ _______, also known as the chain termination method, is a technique for DNA sequencing based upon the selective incorporation of chain-terminating dideoxynucleotides (ddNTPs) by DNA polymerase during in vitro DNA replication

A

Sanger sequencing

223
Q

______ ___: specialized PCR machines with fluorescent detectors for each sample. The product is quantified by:
1. SyBr Green dye: binds dsDNA and fluoresces
2. Primers that fluoresce when bound to template
Product to be quantified is compared against a known control. Sample can be RNA if first reverse-transcribed to a single strand of DNA

A

Quantitative PCR (qPCR)

224
Q

_____ _____: many primers are synthesized with significant overlap by inputting sequence into a computer program. Each primer extends off another by their overlap. More rounds of melting & extension gives longer assembled fragments. Once assembled, start & end primers are used to amplify only the correctly assembled piece

A

Sequence Assembly

BMSC 240 (2 decks)

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