1
Q
- A molecular biology technique which simply makes many
copies of specific DNA or genes which takes place in vitro,
laboratory, or test tube, rather than in the cell.
A
POLYMERASE CHAIN REACTION
2
Q
- PCR relies on thermostable DNA Polymerase, called
A
Taq Polymerase
3
Q
- Heat the reaction strongly to separate, or denature,
the DNA strands.
A
DENATURING
4
Q
This provides single-stranded template for the next step,
annealing.
A
denaturing
5
Q
- DNA synthesis requires that the template DNA be ______
stranded.
A
single
6
Q
- The hydrogen bonds between the complementary bases in the double strands of the template DNA are broken by
heating at what temp??
A
94° to 95°C for 30 to 60 seconds.
7
Q
Cool the reaction so the primers can bind to their
complementary sequences on the single-stranded
template DNA.
A
annealing
8
Q
oTemperature is ?? to allow primers to base
pair to complementary DNA template.
increased or decreased
A
decreased
9
Q
- After denaturation, the temperature is lowered typically
between __°C to __°C for 30 to 60 seconds
A
47°C to 60°C for 30 to 60 seconds
10
Q
The ________ temperature of a DNA molecule is described as the temperature at which half of the DNA strands are in a
single-stranded (ssDNA) state
A
melting
11
Q
- Raise the reaction temperatures so Taq polymerase
extends the primers, synthesizing new strands of DNA.
A
extension
12
Q
The temperature is raised, typically to __°C, to allow the
DNA polymerase to make a complimentary copy of template
starting from the primers.
A
72 deg
13
Q
- A typical PCR incudes _____ cycles of amplification, with a
final extension cycle of 10 minutes at 72°C, followed by
incubation at 4°C.
A
25-40
14
Q
- A typical PCR incudes 25-40 cycles of amplification, with a
final extension cycle of __ minutes at 72°C, followed by
incubation at 4°C.
A
10mins
15
Q
Initial denaturation TEMP/TIME:
A
95°C, 5 min
16
Q
Denaturation TEMP/TIME:
A
94°C, 1 min (25-40 cycles)
17
Q
- It is a technique in a conventional PCR procedure to
visualize or check if the genes are amplified.
A
GEL ELECTROPHORESIS
18
Q
- Gel electrophoresis is a technique in which fragments of
DNA are pulled through a gel matrix by an electric current,
and it separates DNA fragments according to ?
A
SIZE
19
Q
DNA of interest called
A
template DNA
20
Q
It is extracted
from the sample and it contains the target sequence.
A
TEMPLATE DNA
21
Q
Before starting PCR, the purity of the DNA of interest
should be checked using ?
A
spectrophotometer.
22
Q
The higher absorbance means there are ?
A
MORE DNA
23
Q
RATION OF A PURE DNA
A
1.8-2.0
24
Q
RATIO WITH <1.8 MEANS
A
PROTEIN AND SOLVENT CONTAMINATION
25
RATION WITH >2.0 MEANS
rna contamination/ presence of chloroform or phenol
26
* Short single-stranded synthetic oligodinucleotide that
serve to start the DNA synthesis, which are
complementary to the ends of each target DNA.
primer
27
are synthetic oligonucleotides that serve to
initiate DNA synthesis.
primers
28
A pair of primers is required, called ??
forward and reverse
primers
29
* Primers range from ____ nucleotides and are single
stranded.
15-30
30
________ temperature refers to the DNA
molecule
o Melting
31
the temperature level at
which 50% of the DNA template are singlestranded and 50% are double stranded.
melting temperature
32
each primer should not have
complementary bases WITHIN the sequences to
prevent FORMATION OF ?
HAIRPIN LOOP.
33
* Each primer should not have complementary
sequences BETWEEN F primers and R primers will
FORMATION OF ?
PRIMER-DIMER.
34
means accurate synthesis.
high fidelity
35
If there are excess dNTPs, the _____________
will bind to it.
magnesium
36
* building blocks
for the new DNA.
Deoxynucleotide triphosphates (dNTPs)
37
The recommended concentration of each dNTP is ______
uM in each reaction mixture.
200-250
38
Optimum temperature is 72°C
o Makes an error in every 125, 000th nucleotides
o Typically, the recommended concentration in the
reaction mixture is 0.02-0.03 U/uL (unit per
microliter)
THERMUS AQUATICUS
39
o Has proofreading ability
o High fidelity (accuracy) DNA polymerase
o Makes error only in every 676, 000th nucleotid
Pfu DNA Polymerase
o Pyrococcus furiosus
o
40
o It controls the shift of pH by regulating the amount
or the concentration of hydrogen ions in the
solution.
Tris-HCl buffer
41
* Regulates amount of H+ ions in the solution
* Specific pH level: 8.0-9.5
PCR BUFFERS
42
important for DNA Polymerase. It serves
as a cofactor, which is important for the specificity of
annealing of primers.
MAGNESIUM
43
the enzyme of choice for routine
amplification of small segments of DNA. Its optimal
temperature at 72°C.
TAQ POLYMERASE
44
o If there's too much hydrogen ion in the solution, the
hydrogen ion will bind to the phosphate group of
dNTPs leading to the formation of ?
phosphoric acid.
45
* Water used in the reaction mixture must be
?
nuclease-free.
46
a machine that automatically changes
the temperatures and incubation times in each cycle of
the PCR
THERMOCYCLER
47
* The ideal dNTP concentration is ____ µM of each, but
some enzymes may require as much as 400 µM each.
200
48
In dNTPs Lower concentration can _________ enzyme fidelity but the
yield may be reduced.
INCREASE
49
* A method of separating electrically charged substances
in a mixture (e.g., DNA, RNA, proteins, etc.).
electrophoresis
50
* A method of separating electrically charged substances
in a mixture (e.g., DNA, RNA, proteins, etc.).
electrophoresis
51
This electrophoresis technique was first developed by ? in 1930
for the study of serum proteins
Arne Tiselius
52
* A type of electrophoresis in which supporting medium
is a gel
GEL ELECTROPHORESIS
53
ELECTROPHORESIS: 3 PARTS are
voltage, supporting medium, buffer system
54
____________ gel structure held together by covalent
cross-linking of Acrylamide and N, N’-methylene
bisacrylamide
* Tougher than agarose gels
Polyacrylamide Gel (PAGE)
55
* Most effective in separating DNA fragments of
varying sizes from 100bp to 25kb.
what type of gel is this
agarose gel
56
* It is a linear carbohydrate polymer composed of
repeating units of agarobiose, which consists of
alternating units of galactose and 3,6-anhydrogalactose
agarose gel
57
* This is a procedure that separates molecules based on their rate of movement through agarose gel under the influence of an electrical field.
AGAROSE GEL ELECTROPHORESIS
58
* It is placed in the liquid agarose after it has been poured
* Removing the it from the hardened gel produces
a series of wells used to load the DNA.
comb
59
* Its function is to prevent the pH from changing. It is used
to resist pH changes.
running buffer
60
used in analyzing
large sizes (around 1500 bp or higher); generates
less current compared to TBE
tris acetate EDTA
61
used in analyzing
smaller sizes or fragments.
tris borate EDTA
62
* Also known as Molecular
Weight Marker
* It is usually dispensed in
the first lane of the well.
DNA LADDER
63
most used DNA stains
ethidium bromide
64
oDerived from two species of seaweeds: Gelidium
and Glacilaria.
* STANDARD (HIGH-MELTING-TEMPERATURE)
AGAROSES
65
Gel casted horizontally in this gel
agarose
66
this gel is Commonly used for DNA
separations
agarose
67
Running buffer too diluted,
voltage too high results to
a poor resolution
68
* This is used to separate very large DNA molecules.
* This process uses more than one electrical field (e.g., one
direction, horizontal, vertical, or opposite direction).
pulse-field gel electrohporesis
69
: The mobility of DNA molecules is HIGHLY
DEPENDENT ON THE ELECTRICAL FIELD applied to
the gel, because it disrupts hydrogen bonds t or f
true !!
70
a process that involves a
reverse transcriptase (RTase), an enzyme that
uses RNA as the template to make complementary
DNA (cDNA)
reverse transcription
71
primers used in rt-pcr?
random hexamers & oligo dt pimers
72
* This primer is used for templates that does not contain
mRNA. This is used to any RNA template except
mRNA.
* This primer does not require a Poly-A tail in its 3’ end.
RANDOM HEXAMERS
73
* This primer is used if the template is a messenger RNA
(mRNA).
* Oligo dT primers only attach to Poly-A tail, which is why
the sample must have a Poly-A tail in its 3’ end.
* Poly-A tail located in the 3’ end
OLIGO dT PRIMERS
74
a technique used to amplify multiple
target sequences in a single PCR reaction using
multiple primer sets
multiplex-pcr
75
This method is also used to detect different viral,
bacterial and other pathogens in a single tube.
oThe method is commonly used in Microbiology
lab applied in bacteria and viruses.
multiplex-pcr
76
* This method is used to detect deletions,
polymorphisms, mutations, etc.,
multiplex pcr
77
- a modification of PCR that was
designed to improve sensitivity and specificity.
- involves the use of two primer sets
and two successive PCR reactions.
oEXTERNAL/OUTER PRIMERS
oNESTED PRIMERS.
* The process is repeated two (2) times.
nested pcr
78
developed to increase both the
sensitivity and specificity of PCR. This technique uses two
pairs of amplification primers and two rounds of PCR
nested pcr
79
* It is also known as Real-time PCR.
* It monitors the amplification of a targeted DNA
molecule during the PCR (i.e., in real time).
QUANTITATIVE PCR (qPCR)
80
TWO COMMON METHODS FOR THE
DETECTION OF PCR PRODUCTS in real-time
PCR are:
non-specific fluorescent dyes
sequence-specific DNA probes
81
* The ______________ of the real-time PCR reaction refers to the
signal level during the INITIAL CYCLES OF PCR,
usually cycles 3 to 15, in which there is little change in
fluorescent signal
baseline
82
The _______ of the real-time PCR reaction is the level
of signal that reflects a statistically significant
increase over the calculated baseline signal.
threshold
83
* The cycle at which the amplification plot crosses the
threshold (i.e., there is a significant detectable increase
in fluorescence).
* CT can be a fractional number and allows calculation of
the starting template amount
Cycle Threshold
84
* Dictates if the reaction/sample is correct and valid.
* Rerun the sample if it does not contain value for IC.
Internal Control
85
* The time where the target is amplified.
* There is an amplification is increasing.
Exponential Phase
86
* The level of reagent aligns to the target.
Linear Phase
87
* It reaches this phase when the reagents are exhausted
and there are no site for binding of primers.
Plateau Phase
88
The FLUORESCENCE is ? PROPORTIONAL to
the CONCENTRATION of the target.
directly or inveresly
DIRECTLY
89
The CYLE NUMBER (CT Value) is ?
PROPORTIONAL to the CONCENTRATION of the target.
directly or inversely
INVERSELY
90
two types of DNA analysis in qPCR
ds DNA binding dye chemistry
fluorophore-linked probes
91
oIt intercalates with the double stranded DNA and
will form fluorescence. The more concentrated the
target, the more fluorescence.
oThe dye intercalates during extension phase
because dsDNA is only present in extension
phase.
DOUBLE-STRANDED DNA BINDING DYE
92
Unsuitable for high-resolution melt curve analysis (HRM)
SYBR Green
93
* Less inhibitory to PCR;
* Higher amount of concentration will not inhibit the PCR.
* Suitable for qPCR using a fast-cycling protocol
* It is more suitable for high resolution melt curve analysis.
EvaGreen
94
A ?? will result if there are no contaminants
and the result is SPECIFIC.
single peak
95
* A multiple peak will result in the graph if there are
presence of contaminants (e.g., primer-dimer). The first
peak is a?
NON-SPECIFIC PRODUCT.
96
* Their mechanism of action relies on the 5′–3′
exonuclease activity of Taq polymerase, which
degrades the bound probe during amplification.
Hydrolysis Probes
97
Fluorescence will only happen if there is an
_________ in the sequence because that is
where the action of polymerase (exonuclease
activity)
extension
98
enables the absolute and reproducible
quantification of target nucleic acids
digital PCR
99
* It is method is preferred if a person is studying
cancer or mutation because there is partitioning
into multiple chambers.
* Targets will have a fluorescence.
DIGITAL PCR
100
iT is a
specific, simple, rapid and cost-effective nucleic acid
amplification method
Loop-mediated isothermal amplification (LAMP)
101
* It uses 4 to 6 primers (Forward Inner and Outer, Backward
Inner and Outer).
* The reaction time takes about 40-60 minutes. Adding
another set of primers speeds up the reaction to 20-30
minutes.
LAMP
102
This method relies on the auto-cycling strand
displacement deoxyribonucleic acid (DNA) synthesis
which is carried out at a constant temperature water
bath/heat block in the presence of Bacillus
stearothermophylus (Bst) DNA polymerase.
LAMP
103
The process of determining the
sequence of nucleotide bases.
DNA SEQUENCING
104
● This is not a DNA sequencing
technique, but rather a detection
technique.
● It will detect expressed genes.
DNA MICROARRAY
105
Chain Termination Sequencing
SANGER TECHNIQUE
106
Chemical Sequencing
MAXAM-GILBERT:
107
it was the______________
Technique that was used in
1991 during the Human
Genome Project.
Sanger
108
● The target DNA is targeted many
times. It is a PCR based
technique.
SANGER SEQUENCING
109
is a unique component of the
Sanger Technique.
Dideoxyribonucleotides (ddNTP)
110
ddNTP lacks a hydroxyl group
on the _____prime carbon.
THIRD
111
this version requires
chemical modifications of the DNA
and further cleavage and
electrophoresis
MAXAM-GILBERT SEQUENCING
112
A method of sequencing for
single-stranded DNA by taking
advantage of the two-step catalytic
process involving four chemicals
that will selectively attack the
purines and pyrimidines and the
addition of piperidine.
MAXAM-GILBERT SEQUENCING
113
__________-- cuts the DNA strand at
specific locations.
Piperidine
114
In Maxam-Gilbert, For G and A, we add ??
to modify the purines.
formic acid
115
For C and T, we add ? to
break the glycosidic bond between
the ribose sugar and the base
hydrazine
116
__________ and _____ will
selectively cleave cytosine
nucleotides to differentiate it from
thymine.
Hydrazine and salt
117
_____________________
sequencing is the first
technique developed
for DNA sequencing
Maxam-Gilbert
118
________ sequencing
uses radioactively r
fluorescently labeled
ddNTPs.
Sanger
119
what year
● Sanger Technique and
Maxam-Gilbert Technique were
developed.
● These two techniques were
regarded as first generation
sequencing techniques.
1977
120
what year
● The first genome was sequenced.
Human mitochondrial genome
sequence was completed.
● The entire mitochondrial DNA was
sequenced.
1981
121
what year
Complete eukaryotic genome.
1996
122
The Human Genome Project was
completed in the year
2001
123
refers to methods in which millions
of DNA templates are sequenced
simultaneously in a single reaction.
Massively Parallel Sequencing
124
● Are enzymes degrade DNA
molecules by breaking the
phosphodiester bonds that link one
nucleotide to the next in a DNA
strand.
● May be specific for DNA or RNA,
such as DNases or RNases.
nucleases
125
● Hydrolyze internal bonds within or
in between a polynucleotide chain.
ENDONUCLEASES
126
● Remove nucleotides one at a time
from the ends or the external of a
DNA molecule.
EXONUCLEASES
127
● A class of endonucleases able to
cut in between with a restriction
able to recognize a specific site or
restriction site.
RESTRICTION ENDONUCLEASES
128
● Sequences recognized by REs
read the same from left to right as
they do from right to left on the
complementary strand.
PALINDROME
129
___________ occurs in the DNA of the
bacteria and shields it from the
restriction enzymes by not allowing it to
bind.
Methylation
130
● Exhibit both restriction and DNA
modification activities.
● Require the cofactors such as
Mg2+ ions, S-adenosylmethione
(SAM) and ATP for their activity.
TYPE I RESTRICTION ENZYMES
131
If aiming for a specific gene
only, Type I is ideal.
true or false
false! hnd sya ideal
132
● Recognition site is 4-6 bp
sequence, usually palindromic.
○ Fragments produced are
possibly very short.
type II restriction enzyme
133
● Staggered ends on a DNA
molecule with short,
single-stranded overhangs
STICKY ENDS
134
● Have straight cut down through
the DNA, that results in a flat pair
of bases on the ends of the DNA.
. BLUNT ENDS
135
● Possess both restriction and
modification activities (same with
Type I).
● Recognizes specific sequences
and cleaves 25-27 bp outside of
the recognition sequence, in a 3’
direction (asymmetrical).
● Requires 2 restriction sites in
opposite orientation.
Requires Mg2+ ions for their
activity.
TYPE III RESTRICTION ENZYMES
136
used for Correct ionic strength
NaCl or KCl
137
If the fragmented DNAs is
asymmetrical, it will have to
undergo _____________ in order to
know what base pair it enters.
electrophoresis
138
If the fragmented DNAs is
asymmetrical, it will have to
undergo _____________ in order to
know what base pair it enters.
electrophoresis
139
a circular DNA
found in bacteria that is
used in gene cloning. This
is the DNA the bacteria
uses for antibiotic
resistance.
plasmid
140
● One of the simplest methods in
identifying mutations.
● Distinguish gene alleles by
specifically recognizing single
base changes in DNA.
SINGLE NUCLEOTIDE
POLYMORPHISMS (SNP)
141
● This is a method used to map an
unknown segment if DNA by
breaking it into pieces and then
identifying the location of the
breakpoint.
● The positions of the sites can be
inferred based on the sizes of the
resultant DNA fragments.
RESTRICTION ENZYME DNA
MAPPING
142
Genetic alteration that
contributes to complex
disease has smaller effect
polymorphism
143
* PYRIMIDINE TO PURINE
* T-A to G-C
transversion
144
* Occurs internally
* This type of mutation is inevitable and uncontrolled.
SPONTANEOUS MUTATION
145
* Occurs externally
* This type of mutation is controlled
INDUCED MUTATION
146
* A change in a single nucleotide in the genome that causes
variations in DNA sequences between members of the same
species.
* Occurs when two individuals in the population differ by a
single base in the DNA sequence.
* Can act as biological markers.
SINGLE NUCLEOTIDE POLYMORPHISM (SNP)
147
* Caused by a single gene, several different mutations
* Can result in the same disease but with varying degrees
of severity and phenotype
SINGLE GENE DISORDER
148
* Main factor is genes but the cause includes other factors
that aren’t genes
MULTIFACTORAL DISORDER
149
* A technique that is used to study genetic variation or
polymorphisms among individuals using restriction
enzymes.
CLEAVAGE BASED: Restriction Fragment Length
Polymorphism (RFLP)
150
After heating and cooling the mixture, which of the following catalyzes the synthesis of complementary strands of DNA?
taq pol
151
DNA is visualized by including in the gel an intercalating dye is:
a.ethidium bromide.
b.methylene blue
c.basic dye
d.eosin dye
a
152
Gel electrophoresis separates DNA fragments by size in a solid support medium called as:
agarose gel
153
Pfu polymerase is superior over Taq polymerase because:
of more accurate amplification
154
Polymerase used for PCR is extracted from _____________.
thermus aquaticus
155
A method that requires a set of four specifically designed primers that can recognize six distinct regions on the target DNA
lamp
156
A technique used to amplify multiple target sequences in a single PCR reaction using multiple primer sets.
multiplex PCR
157
An enzyme that uses RNA as the template to make complementary DNA (cDNA)
REVERSE TRANSCRIPTASE
158
Fidelity in amplification may be decreased in which of the following conditions?
Increased concentrations of dNTPs
159
It is adjusted to a value above the background of the reaction
THRESHOLD
160
Low to no yield of PCR products may be a result of which of the following
Decreased MgCl2 and Decreased dNTPs
161
Small fluorescent molecules that are attached to oligonucleotides in order to function as probes
FLUOROPHORES
162
Their mechanism of action relies on the 5′–3′ exonuclease activity of Taq polymerase
HYDROLYSIS PROBE
163
This PCR technique was designed to to improve sensitivity and specificity.
NESTED PCR
164
Which of the following PCR techniques is the gold standard for testing COVID-19 infection?
RT-PCR
165
To sequence DNA by Sanger method, the DNA must first be made in single stranded form.
T OR F
TRUW
166
When a dideoxyribonucleotide is added to the DNA strands being synthesized:
replication of the strand stops
167
To sequence DNA by Maxam-Gilbert method, the DNA must first be made in single stranded form.
TRUE OR F
TRUE
168
What is the difference between a deoxyribonucleotide and a dideoxyribonucleotide?
a. A deoxynucleotide is missing a 5'-phosphate group.
b. A deoxynucleotide is missing a 3'-hydroxyl group on its sugar.
c. A dideoxynucleotide is missing a 5'-phosphate group.
d. A dideoxynucleotide is missing a 3'- hydroxyl group on its sugar.
d. A dideoxynucleotide is missing a 3'- hydroxyl group on its sugar.
169
ATP, Mg2+ WHAT TYPE OF RESTRICTION ENZYME
TYPE III
170
Cleave within or at a short distance from the recognition site
TYPE II
171
Cleaves at sites away from recognition site
TYPE I
172
Cleave close to or within recognition sequence
TYPE IV
173
Are endonucleases that acts as a Molecular
scissors that cut double stranded DNA molecules at
specific points also known as restiction sites
RESTRICTION ENZYMES
174
is a virus that infects bacteria
in the process called transduction
Bacteriophage
molbio
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