NUCLEIC ACID AMPLIFICATION TECHNIQUES Flashcards
1
Q
the very first amplification method
A
PCR - polymerase chain reaction
2
Q
Nucleic acid (NA) amplification methods fall
into three categories:
A
target amplification
probe amplification
signal amplification
3
Q
Involves making copies of a target
sequence to such a level (in millions
of copies) that they can be detected
in vitro.
A
target amplification
4
Q
This is analogous to growing cells in
the culture and allowing the cells to
replicate their nucleic acids, as well
as themselves
A
target amplification
5
Q
The difference between
microbiology and molecular
biology amplification is
A
in microbiology - amplification is days to months
in target amplification minutes to hours
6
Q
–The first prototypical (specific)
method for amplifying target nucleic
acids.
A
polymerase chain reaction
7
Q
PCR advantages
A
user friendly
more amendable
automated
8
Q
gold standard in the detection of covid 19
A
rt PCR
9
Q
1983, ___ conceived the idea of amplifying dna in vitro
A
kary mullis
10
Q
–In the process of working through a
mutation detection method, he
came up on a way to double the test
targe
A
kary mullis
11
Q
PCR Initially used the “_”
method.
A
3 graduate student
12
Q
Knew that you could use __ to initiate DNA
synthesis
A
primers
13
Q
first sample to be used in amplification used by kary mullis
A
nerve growth factor
14
Q
the first successful sequence to amplify is
A
short fragment of escherichia coli
pBR322
15
Q
PCR is called previously as
A
Polymerase Catalyze
Chain Reaction
there’s catalyze because there’s dna polymerase used before
16
Q
A
17
Q
A
18
Q
A
18
Q
A
18
Q
A
18
Q
A
18
Q
A
19
Q
A
19
Q
REQUIREMENTS for PCR
A
- double stranded DNA - template - to give the order of nucleotide
- Deoxyribonucleotide bases
- 1 enzyme for addition of nucleotide to growing strands - DNA polymerase
- primer
20
product of PCR -
amplicons
21
* One copy of dsDNA becoming two copies.
In vivo
* About an hour or two, PCR can produce millions of
copies called amplicons of DNA
in vitro PCR
22
* In contrast, it would probably take several days for
a cell to produce the same number of copies in vivo.
in vivo replication
23
3 graduate steps in pcr
denaturation
annealing
extension
24
temp and time for denaturation
90-96 * C
20-60secs
25
temp and time for annealing
50-70 *C
20-90 secs
26
temp and time for extension
68-75*C
10-60 secs
27
target in pcr
double stranded DNA
28
a process wherein we Reduce double stranded molecules to single stranded molecules
denaturation
29
This is accomplished by heating the samples by 90 or 94-96 *C for 20-60 seconds up to several minutes, depending on the type
of target for testing
denaturation
30
the most critical step in specificity of the pcr
annealing
31
In this step, there are two oligonucleotides, which will prime the
synthesis of DNA and will anneal to the complementary sequence of the
template.
annealing
32
__determines the specificity of the amplification.
Primer
33
The __ dictates the part of the template that would be amplified.
primer
34
the location where will the primer attach during PCR
oligonucleotides
35
what are the 2 types of primers
forward and reverse
36
the type of primer that will hybridize to the complementary strand just below the 5 prime
forward primer
37
the type of primer that hybridizes to the 3 prime of the sequence to be amplified
reverse primer
38
EXTENSION (PRIMER EXTENSION) also called as
elongation
39
This is when the DNA synthesis occurs.
EXTENSION
40
The DNA Polymerase catalyzes the formation of the phosphodiester bonds
in between the incoming DNTPs which is then determined by the hydrogen
bonding to the template.
EXTENSION (PRIMER EXTENSION)
41
bases always attach to which part of the primer
3` end
42
In some cases, the
annealing temperature
is close enough to the
extension temperature
that the reaction can
proceed with only 2
temperature changes.
Two-Step PCR
43
COMPONENTS OF PCR
1. Primers
2. DNA template
3. Deoxyribonucleotide
Bases
4. DNA Polymerases
5. PCR Buffer
6. Thermal Cyclers
7. Controls for PCR
44
Critical component of PCR because they determine the
specificity of the PCR
primer
45
primer
They are analogous to the _ in blotting and hybridization procedures
probes
46
primers are desifgned using genomic sequence available from
national center for biological information
47
factors that affect primers
1. primer sequence - number of guanine and cytosine - must be a lot
2. length affecting the conditions in which the primer will bind to its target
48
the 2 factors that affecr primer is affected by ___
the melting temperature of the primer
49
served as the starting point in setting optimal annealing - starting point for optimal binding
melting temperature
50
primers (forward) its melting temp can be adjusted in the length of the template and number of guanine and cytosine
t or f
true
51
primers
single stranded DNA fragments that how many bases in length
20-30 bases in length
52
problems in primer
mispriming
primer dimers
53
creation of unintended product caused by primers attaching to wrong area
mispriming
54
how to prevent mispriming
HOT start PRC
55
PCR products that are double the size if the primer, results from the binding of primer with each other (on the 3` end)
primer dimers
56
it connects all bases to each other
phosphodiester bond
57
connects the primer to template
hydrogen bond
58
Clinical sample sources of dna template
all microorganism
59
what kind of sample if we are using hair follicle
mitochondrial DNA
60
PCR reagents and conditions, nanogram amounts of genomic DNA are
sufficient for consistent results
true or false
true
61
how many data template are we using?
❑Routine clinical analysis - 100 ng to 1 μg of
DNA is usually used
62
Lesser amount of data template are required for more
defined template preparations such as
_____ DNA or product from a
previous amplification
cloned target
63
best templates
good condition
free of contaminating proteins
without nicks or breaks that can stop DN synthesis or cause misincorporation of nucleotide bases
64
templates with high GC content and secondary structure may prove more ____ to optimize for amplification
difficult
65
are
the building blocks of DNA
Nucleotide triphosphates
66
Deoxyribonucleotide
triphosphates (dNTPs
1. Adenine
2. Thymine
3. Guanine
4. Cytosin
67
In common PCR applications,
the recommended final
concentration of each dNTP is
generally
0.2 mM
68
why does the building block of the DNA, nucleotide triphosphate, must be a triphosphate and not a mono or di-
diphosphate results from poor manufacturing conditions and mono and diphosphate are contaminated with heavy metals
69
during the PCR, the dNTPs function as the nucleotide of a growing strand of DNA. They are added to the growing strand with the enzyme ___
Taq DNA polymerase
70
DNA polymerase, kary mullis first performed PCR, he used the DNA polymerase isolated from ___
E coli
71
first molecule of the year 1985
Taq polymerase - enzyme of PCR
72
Taq polymerase is isolated from
thermus aquaticus
73
Using an enzyme derived from a thermophilic
bacterium meant that the DNA polymerase could be added once at the beginning of the procedure and it would maintain its activity throughout the heating and
cooling cycles
true or false
true
74
an enzyme derived from thermophilic bacterium that Were subsequently exploited for laboratory use
Tth polymerase
75
an enzyme derived from thermophilic bacterium that has reverse transcriptase activity so that it can be used in reverse transcriptase PCR OR rt-pcr
tth polymerase
76
an enzyme derived polymerase that have proof-roofing functions and can generate products over 30kbp (30, 000 bases in length)
vent polymerase
77
Taq polymerase is first isolated in year ___ at ____
1969 at yellow stone national park in california
78
modified version of Taq polymerase
stoffel fragments
79
stoffel fragments
modified version of taq polymerase that lack amino acid on the __- terminal postion of taq polymerase
289th terminal
80
advantage of stoffel fragments
has 3` to 5` exonucleases
81
half life of stoffel fragments
double in high temp of that of Taq polymerase
82
purpose of stoffel fragments
alleles specific PCR and for amplication with high guanine and cytosine contents
83
provides optimal conditions for enzyme activity
pcr buffer
84
PCR BUFFR
The buffer pH is usually between ___ and is often stabilized by Tris
HCl.
8.0 and 9.5
85
monovalent cations example
potassium chloride
ammonium sulfate
86
divalent cation
magnesium chloride
87
these affects the annealing and denaturation temperature of DNA and enzyme activity
monovalent cation
88
it affects the primer annealing and is very important for enzyme activity
divalent cation
89
this will lower the amount of magnesium available for the enzyme
EDTA or other chelators
90
Too few Mg2 ions wil cause ___ to enzyme
lower enzyme efficiency resulting in a low yield of PCR product
91
overly high Mg2 concentrations will cause ___ to enzymes
promote misincorporation and thus increase the yield of nonspecifc products
92
lower Mg2 concentrations will cause ___ to enzymes
this concentration is desirable when fidelity of the PCR is critical
93
recommended range of MgCl2 concentration is ____
1-4 mM
94
range of MgCl2 in standard reaction conditions
1-4 mM
95
Tris buffer concentration ____ and accessory buffer components are also important for optimal enzyme activity and accurate amplification of the intended product
10 mM
96
purpose of accessory components
used to optimize reactions
97
an accessory component that binds inhibitors and stabilizes the enzyme
bovine serum albumin (10-100 ug/ml)
98
an accessory component that provides reducing conditions that may enhance enzyme activity
dithiothreitol (0.01mM)
99
an accessory component that is added to the reaction mixture and will lower the denaturing temperature of DNA with high secondary structure, thereby increasing the availability for primer binding
formamide (1-10%)
100
101
substitute for dithiothreitol
beta 2 mercaptoethanol
102
aside from the accessory components we tackle about, we can add ___ as well to allow polymerase extensions in difficult aea
GITC guanidine isothiocyanate
triton X100
glycerol
dimethyl sulfoxide
103
