Final Exam (cumulative) Flashcards
(224 cards)
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
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
3
Q
What is sonication?
A
Using sonic/sound/vibration to rip cells apart; vibration also makes heat
4
Q
What is the French Press?
A
Using pressure to “pop” the cells open
5
Q
What is Osmotic Shock?
A
Low salt solution, this is not effective for regular cells walls, but is used for RBCs
6
Q
What is Digestion?
A
Using enzyme digests
7
Q
What are Detergents?
A
Break up fats and oil, break up the phospholipid bilayer
8
Q
What is Homogenization?
A
Using brute force, like a blender/grinder for bigger chunks of solids
9
Q
Proteins removed from a cell degrade by the non-_______ conditions
A
physiological
10
Q
In extract stabilization, care should be taken to avoid 4 things: ?
A
- pH changes
- Heat denaturation
- Oxidation
- Proteolysis
11
Q
What would solve the issue of pH changes in extract stabilization?
A
Extract/keep in buffered solutions
12
Q
What would solve the issue of pH changes in heat denaturation?
A
Keep sample cold, heat only as needed
13
Q
What would solve the issue of pH changes in oxidation?
A
Reducing agents can be added to prevent spontaneous crosslinking
14
Q
What would solve the issue of pH changes in proteolysis?
A
Lower temperature, or add protease inhibitors
15
Q
What are proteases?
A
Enzymes that break down proteins, proteolysis
16
Q
To separate the protein/enzyme you want from everything else you have to be able to track it using an ____
A
assay
17
Q
Assays should be
– _____
– _____
– ____
– ______
A
Sensitive, Specific, Rapid, Quantitative
18
Q
What is Activity in terms of Purification Tracking?
A
how many U of enzyme per mL
19
Q
What is Specific Activity in terms of Purification Tracking?
A
how many U per mg protein
20
Q
What is Yield in terms of Purification Tracking?
A
(total Activity after purification) / (total Activity before
purification)
21
Q
What is Purity in terms of Purification Tracking?
A
(S.A. after) / (S.A. before)
22
Q
What is a method based in Solubility for Purification by physical properties?
A
Salting out
23
Q
What are 3 methods based in Charge for Purification by physical properties?
A
Ion exchange chromatography
Electrophoresis
Isoelectric focusing
24
Q
What is a method based in Polarity for Purification by physical properties?
A
Reverse phase (hydrophobic) chromatography
25
What are 3 methods based in Size for Purification by physical properties?
Dialysis
Size-exclusion chromatography
Gel electrophoresis
26
What is a method based in Binding Specificity for Purification by physical properties?
Affinity chromatography
27
What is a method of initial purification?
Centrifugation
28
If the protein is soluble it can be partially purified by removing larger/denser contaminants by differential _______
centrifugation
29
In centrifugation, the faster the spin, the _____ the contaminants can be removed
smaller
30
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 ___
less, temperature, salinity, pH
31
As [salt] _____, the + & - ions will compete with the hydrophilic surface amino acids for water the protein molecules with insufficient hydration will aggregate & lose _____
increases, solubility
32
In dialysis, excess salt must be ______ as it reduces solubility and can impede subsequent isolation steps
removed
33
Dialysis tubing has pores with a specific molecular
weight cut-off that allow _____ molecules (salt) to pass.
smaller
34
Dialysis is great for separating proteins from salts, but not for separating proteins themselves based on ____
size
35
________ is a technique used to separate mixtures based on physical properties such as size or charge
Chromatography
36
What are the two phases in chromatography?
Stationary and mobile
37
What is the stationary phase in chromatography?
A substance that the compounds to be separated pass by or interact with
38
What is the mobile phase in chromatography?
The carrier for the compounds to be separated
39
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
variable, mobile, stationary
40
With different affinities in chromatography come different rates of migration = ?
separation of the molecules
41
What are the 5 basic steps in column chromatography?
1. Pouring
2. Packing
3. Loading
4. Running/eluting
5. Collecting
42
In what kind of chromatography do the porous beads make up the stationary phase?
Size exclusion
43
In size exclusion chromatography, there are several important volume concepts:
– Vt:
– Vo:
– Vi:
– Ve:
The volume of the column
The volume outside the beads
The volume inside the beads
The volume at which a sample is eluted
44
In size exclusion chromatography, _____ proteins can not enter the beads so they will migrate around them so they travel in only the ___
large, Vo
45
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 ___
moderate, Vo, Vi
46
In size exclusion chromatography, small proteins do not interact with the beads and move the slowest so there is no Vo or Vi, just ____
Vt
47
The _______ _______ is the fraction of the pores within the beads available to the sample
partition coefficient
48
Kav = __ = always excluded by beads
Kav = __ = beads fully accessible
0, 1
49
What is the formula for Kav?
Kav = (Ve – Vo)/(Vt - Vo)
50
Partition coefficient can be used to estimate ?
molecular weight
51
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
length, shape
52
If Kav is >0.8 or <0.2 you will get ____ separation of proteins
poor
53
Samples become diluted as:
______ from layering sample over gel
______ during travel
______ differences along the glass tube wall
Turbulence, Diffusion, Friction
54
A column of positively or negatively charged beads forms the stationary phase.
_____ exchange (column is +, targets are -)
_____ exchange (column is -, targets are +)
Anion, Cation
55
In ion exchange chromatography, proteins in solution get carried down by gravity and will get slowed by the beads proportionally based on _____
charge
56
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
2, focusing
57
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
cleared bacterial lysate
58
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
affinity
59
Order the 3 kinds of chromatography from most to least specific: ?
1. Affinity chromatography
2. Ion exchange chromatography
3. Size exclusion chromatography
60
_______: migration of ions in an electric field
Electrophoresis
61
In electrophoresis, velocity is directly proportional
to ____ and inversely proportional to ____ and ___
charge, size, shape
62
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
protein
63
A gel is used to ?, ?, and _____ ______
slow down movement, prevent diffusion of the proteins, create separation
64
What does SDS-PAGE stand for?
Sodium dodecyl sulfate polyacrylamide gel
electrophoresis
65
SDS = a _____ that will denature proteins and give them a negative charge
detergent
66
~____ g SDS / ___ g protein
1.4, 1.0
67
Monomers are ______, _______ and ______ , but once polymerized it’s safe
neurotoxic, carcinogenic teratogenic
68
Acrylamide (acrylic amide) = a plastic ______
monomer
69
Acrylamide: forms ____
chains
70
Bis-acrylamide: branch _____/_______
chains, crosslinking
71
Acrylamide + catalyst + a suitable buffered salt solution = _____
gel
72
Around a __:__ mix of acrylamide to bisacrylamide
30:1
73
What are the 5 basic steps in the SDS-PAGE procedure?
1. Casting the gel
2. Forming the wells with a comb
3. Preparing the gel
4. Running the gel
5. Visualizing the gel
74
Protein samples are boiled for __ minutes in __x loading buffer
5, 5
75
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
10, 20, 0.2, 0.05, 10
76
In the SDS-PAGE procedure, there are 3 different dyes that can be used, what are they?
1. Coomassie blue (fastest and least sensitive)
2. Silver nitrate (most complicated, most sensitive)
3. Fluorescent dye staining (sensitive, requires special equipment)
77
Describe the protocol of silver staining: ?
Requires more than half a dozen unique solutions to turn a gel into a piece of black & white photographic film
78
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
bigger, smaller, ladder
79
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
lower, higher
80
The height of a well can produce a vertically diffuse band, there are 4 solutions to this: ?
1. Use less volume per sample
2. Concentrate the sample
3. Run a vertically longer gel
4. Run a stacking gel
81
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?
The bands will be faint
82
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?
Much more time consuming
83
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?
More time consuming, requires a non-standard apparatus
84
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?
Required a special 2-layer gel
85
2 gels + 2 buffers + 3 pH’s = _____ solution
elegant
86
Heating and running samples in a gel made with ~6 M urea allows for proteins to denature but retain their _____ _____
native charge
87
In _____ gels, mobility of proteins depends on size, charge and conformation (shape), and can be used to keep protein complexes intact
native (Non-denaturing)
88
In native gels, charge depends on the ___ of the buffer used
pH
89
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
native
90
____ hemoglobin has a higher affinity for oxygen than _____
fetal, adult
91
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
2-D protein
92
In 2-D protein electrophoresis, what is Dimension 1?
Isoelectric Focusing Gels
93
_____ are molecules that can buffer at their pI
Ampholytes
94
______ _____ 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
Isoelectric Focusing
95
In ______ _____ gels, proteins are electrophoresed
through the ampholyte-containing gel until they hit their pI, where they become uncharged and stop
Isoelectric focusing
96
In 2-D protein electrophoresis, what is Dimension 2?
SDS-PAGE
97
In _____ gels, isoelectric Focusing Strips are treated to denature proteins and coat with SDS. Now-uniformly-negative SDS+proteins are next resolved by size
SDS-PAGE
98
What kind of gel can rapidly separate thousands of proteins in a mixture to individual spots?
2D
99
What kind of gel can identify samples from different sources can be compared to determine what proteins are different?
2D
100
What kind of gel can have spots that can be excised and identified if necessary?
2D
101
_____ as a group they recognize antigens, but specifically they each bind a single epitope
Antibodies
102
Mammals make 5 varieties of antibodies that vary by their tail (immunoglobulins): ?
1. IgG
2. IgM
3. IgA
4. IgD
5. IgE
103
What is an IgG?
Gamma globulin
104
___ are monomers, bivalent, and are about 80% of serum antibodies.
IgG
105
___ recruit the immune system, are in the blood, lymph, and intestine, and can cross the placenta
IgG
106
____ enhance phagocytosis; neutralize toxins and viruses; protects fetus and newborn, and has a half-life of 23 days
IgG
107
Immature B cells have several __, __ and __ DNA sequences – Like having multiple items of pants, shirts & shoes to make up a diversity of outfits
V, D, J
108
Random _____ in the light and heavy chains make unique antibodies
deletions
109
In B-Cell clonal selection, there are 2 tests:
Test #1 – ?
If yes, kill cell
Test #2 – ?
does the B-cell make host binding antibodies?
does the B-cell make useful antigen binding antibodies?
110
Naïve B-cells will be activated upon antigen exposure to form ______ ___ or _____ _____
plasma cells, memory B-cells
111
Antibody ____ rises and falls
titer
112
______ boosts the amount of antibodies produced. Memory B-cells will retain specific Ab potential for a ____ (or less)
Reinfection, lifetime
113
What are the 5 protective mechanisms of binding antibodies to antigens in "in vivo"?
1. Neutralization
2. Agglutination
3 Opsonization
4. Activation of complement
5. Antibody-dependent cell-mediated cytotoxicity
114
What is neutralization?
Blocks adhesion of bacteria and viruses to mucosa; blocks attachment of toxin
115
What is agglutination?
Reduced number of infections units to be dealt with
116
What is opsonization?
Coating antigen with antibody enhances phagocytosis
117
What is Activation of Complement?
Causes inflammation and cell lysis
118
What is Antibody-Dependent Cell-Mediated Cytotoxicity?
Antibodies attached to target cell cause destruction by eosinophils and NK cells
119
Secondary antibodies can be produced in a different organism using the tail (Fc) from _____ antibodies
primary
120
Tails are shared similarity by _____, humans are the same, but not compared to hamster, etc
species
121
______ 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
Polyclonal, Monoclonal
122
In ______ production, cells fused with cancerous B-cells to make them immortal, metabolic & immunological selection for fused cells. The result is called a hybridoma
monoclonal
123
_____ Blotting: a method to look for a protein of interest from a gel using antibodies against the protein
Western
124
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
Western Blotting
125
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 ?
detect the presence of the 1° antibody
126
What are the 4 types of antibody labels?
1. Isotope labelling
2. Enzyme labelling
3. Fluorescent labelling
4. Heavy element labelling
127
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
Isotope Labelling
128
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
Enzyme Labelling
129
What kind of antibody labelling is described?
A dye is attached that absorbs one color of light and gives off another
Fluorescent labelling
130
What kind of antibody labelling is described?
Metals or rare earth elements that are detectable by electron microscopy
Heavy element labelling
131
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
Western Blot
132
What does ELISA stand for?
Enzyme-linked-immunosorbent assay
133
ELISA steps similar to western blots, but with no ______ _____
protein separation
134
What is the difference between an Antigen and an Epitope?
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
? 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
Rapid strip tests
136
________ test are used to test cell specimens, but especially bacterial and blood cells
Agglutination
137
In an ________ test, an emulsion of cells is added to an antibody mixture and rocked back and forth
agglutination
138
Agglutination tests can be done in 2 ways: ?
Unknown cells with specific antibody
Known cells with unknown serum
139
The precipitin Reaction and the ouchterlony assay are both under the category of what kind of assay?
Diffusion
140
_______ assays are useful when samples are too small to agglutinate visibly
Diffusion
141
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
Diffusion assay
142
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
Immunofluorescent Staining + Fluorescent Microscopy
143
What are the 3 kinds of fluorescent microscopy?
1. Standard
2. Confocal
3. Two-photon
144
What is Standard fluorescent microscopy?
Whole image is illuminated and out-of-focus light makes the in-focus image fuzzy
145
What is Confocal fluorescent microscopy?
Whole image is illuminated and out-of-focus light is blocked
146
What is Two-photon fluorescent microscopy?
Less energetic light is used (infrared) and 2 photons must hit the dye simultaneously to excite it
147
What are the pros and cons of Standard fluorescent microscopy?
Pro: cheap
Con: fuzziness and photodamage
148
What are the pros and cons of Confocal fluorescent microscopy?
Pro: very good resolution
Con: photodamage
149
What are the pros and cons of Two-photon fluorescent microscopy?
Pro: lower photodamage, better tissue penetration
Con: costliest, inferior resolution to confocal
150
Which of the 4 nitrogenous bases are purines?
Adenine and guanine
151
Which of the 4 nitrogenous bases are pyrimidines?
Thymine and Cytosine
152
The duplex of DNA is held together by _____ bonding and ____ ______ forces
hydrogen, base stacking
153
What are the 5 different plasmid types?
1. Conjugative (F)
2. Resistance (R)
3. Colicinogenic (Col)
4. Degradative
5. Virulence (vir)
154
What are Conjugative plamids (F)?
Transmitted during conjugation, carry a variety of information
155
What are Resistance plamids (R)?
Protect against environmental factors, MDR (multiple drug resistance) plasmids
156
What are Colicinogenic plasmids (Col)?
Codes for proteins that kill other microbes
157
What are Degradative plamids?
Contain genes for novel catabolic enzymes
158
What are Virulence plamids (vir)?
Increases the pathogenicity of a bacteria
159
LF+PA = a ____ toxin which kills human cells
lethal
160
EF+PA = _____ toxin which causes cells to leak
cytoplasm
edema
161
Cap(A-E) = ____ formation to avoid immune system detection
capsule
162
A _____ is a plasmid that has been streamlined and modified to make it amenable to carrying a payload of DNA
vector
163
Here are the typical components of a cloning vector:
– _____ of replication
– _______ selection gene
– Insert _______ gene
– _____ sites (places to insert foreign DNA)
Origin, Positive, differentiation, Cloning
164
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
3, 1, 4, 5, 2
165
What is the R/M system?
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
What are the 3 types of Restriction Enzymes?
1. Type I
2. Type II
3. Type III
167
Type ___ restriction enzyme: R+M - a specific sequence is recognized by a multi-subunit protein, a random sequence is cut hundreds of bases away
I
168
Type ___ restriction enzyme: R - a specific sequence is recognized (4-8 bp palindrome) by a single protein and is cut within that sequence
II
169
Type ___ restriction enzyme: R - a specific sequence is recognized by a multi-subunit protein, a random sequence is cut ~25 bp away
III
170
What is the difference between sticky and blunt ends of restriction enzymes?
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
_____ digest: reactions can use 2 enzymes at once
Double
172
If a double digest is incompatible, digests can be
done sequentially: ?
reaction 1 → stop (heat denature enzyme) →
change buffer → reaction 2
173
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)
top, distances, different
174
DNA + salt solution + electricity = DNA pulled to ?
Cathode (+)
175
In an agarose gel, the gel immersed in buffer, what are the 3 most commonly used?
TAE, TBE, SB
176
_______ Agent: will be flat and insert itself into the laddered runs of DNA in between the base pairs
Intercalating
177
When we want to visualize the DNA:
_______ ______ + DNA + UV light → orange bands
____ _____ + DNA + blue light → green bands
Ethidium bromide, SyBr Green
178
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
linear, faster, slower
179
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
tumble, reptate
180
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
lower, higher
181
What is Star Activity?
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
How do we solve the issue of having fragments that are too large?
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
We can cut bacterial chromosomal DNA w/ rare enzyme and compare fragments to create ?
strain → specific patterns, a unique “fingerprint” for that DNA
184
____ is an enzyme which connects the phosphodiester backbone of DNA between a 3’-OH and 5’-P
Ligase
185
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
ligase
186
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
E. coli, 42, 30-90
187
A colony on a plate = one _______ event
transformation
188
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
pressure
189
What are the 4 most common antibiotics?
Ampicillin, Kanamycin, Tetracycline, Chloramphenicol
190
What are the 5 steps in a miniprep?
1. Extracting plasmid
2. Resuspended
3. Lysed by NaOH, SDS, boiling
4. Precipitate debris – neutralize NaOH
5. Spin out debris
191
Similar to protein extractions: use _____, _______, ____ to break apart cells
chemicals, temperature, force
192
Solvent extraction needs ______/______ to remove contaminants; and differential precipitation using _____ and _____
phenol/chloroform, salts alcohol
193
? : is a method used to allow studying DNA in an un-cloned state by selective copying and amplification
PCR
194
The basic components if the PCR mechanism include 5 things: ?
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
What is the formula for PCR, and what does each components signify?
of copies = (2^n– 2n)x
Where n = # of cycles
x = # of copies to start
196
What are the 3 basic steps in the first step of PCR?
Melt/denaturation: 94-95°C for 30-120s
Anneal: 45-65°C for 30-120s
Extend: 72°C
197
In the PCR reaction mixture, there is a large range of template DNA, ranging from 10pg - 1µg, why is this?
Because a plasmid can equal one copy of sequence, a genome can equal one copy of the sequence
198
What does PCR stand for?
Polymerase Chain Reaction
199
Amplification will ______ as template binds template instead of primer at later cycles of PCR
plateau
200
_____ polymerase: first discovered (cheapest), 5’ to 3’ polymerase without 3’ to 5’ exonuclease
Taq
201
____ polymerase: somewhat costlier, but more processive (longer pieces can be amplified), fixes errors 25X more often (3’-5’ exonuclease), no 3’ A overhangs
Pfu
202
__-__ bp is optimal for a primer
18, 24
203
_____: avoid repeated sequences, ~50% GC, GC 5’ end clamp, pairs +/- 2°C, avoid hairpins and primer dimers
Primers
204
______ are essential with such a sensitive method to amplify DNA, _____ ones are the easiest to do
Controls, negative
205
What are the 3 kinds of thermocyclers/PCR machines?
Basic, gradient, real-time
206
What is a Basic thermocycler?
Every tube cycles together
207
What is a Gradient thermocycler?
Allows optimization of annealing temps
208
What is a Real-Time thermocycler?
Allows quantification of each cycle
209
____ is the temperature that a double stranded sequence of the primer would fully melt into single stranded molecules
Tm
210
Annealing temperature of a PCR cycle is generally initially tested as 5°C below ___
Tm
211
What is the original formula for calculating Tm?
Tm = 2°C x (A+T) + 4°C x (G+C)
212
What is the problem with the original formula for calculating Tm?
Does not have an upper limit, so it is only valid for about 20bp
213
What is the benefit for the more complicated formula for Tm?
Takes into account the salt concentration and order of the bases
214
PCR is sensitive enough that you can amplify false positives because of contaminating DNA from: ?
You
Your pipette
The water
Other random errors
215
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
repeated, unique
216
What does CODIS stand for?
Combined DNA index system
217
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
13, meiosis, phenotypes, inherit
218
CODIS Markers can identify ______ individuals
Unique
219
Blood can be used for exclusion, we can determine it (may/may not?) be you
may not
220
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
disease, amplify
221
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
single,
222
_____ _______, 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
Sanger sequencing
223
______ ___: 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
Quantitative PCR (qPCR)
224
_____ _____: 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
Sequence Assembly
