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MODX > NUCLEIC ACID AMPLIFICATION TECHNIQUES > Flashcards

NUCLEIC ACID AMPLIFICATION TECHNIQUES Flashcards

1
Q

the very first amplification method

A

PCR - polymerase chain reaction

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2
Q

Nucleic acid (NA) amplification methods fall
into three categories:

A

target amplification
probe amplification
signal amplification

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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

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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

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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

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6
Q

–The first prototypical (specific)
method for amplifying target nucleic
acids.

A

polymerase chain reaction

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7
Q

PCR advantages

A

user friendly
more amendable
automated

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8
Q

gold standard in the detection of covid 19

A

rt PCR

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9
Q

1983, ___ conceived the idea of amplifying dna in vitro

A

kary mullis

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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

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11
Q

PCR Initially used the “_”
method.

A

3 graduate student

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12
Q

Knew that you could use __ to initiate DNA
synthesis

A

primers

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13
Q

first sample to be used in amplification used by kary mullis

A

nerve growth factor

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14
Q

the first successful sequence to amplify is

A

short fragment of escherichia coli

pBR322

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15
Q

PCR is called previously as

A

Polymerase Catalyze
Chain Reaction

there’s catalyze because there’s dna polymerase used before

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16
Q
A
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17
Q
A
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18
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A
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18
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18
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18
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18
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19
Q
A
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19
Q

REQUIREMENTS for PCR

A
  1. double stranded DNA - template - to give the order of nucleotide
  2. Deoxyribonucleotide bases
  3. 1 enzyme for addition of nucleotide to growing strands - DNA polymerase
  4. primer
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20
Q

product of PCR -

A

amplicons

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21
Q
  • One copy of dsDNA becoming two copies.
    In vivo
  • About an hour or two, PCR can produce millions of
    copies called amplicons of DNA
A

in vitro PCR

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22
Q
  • In contrast, it would probably take several days for
    a cell to produce the same number of copies in vivo.
A

in vivo replication

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23
Q

3 graduate steps in pcr

A

denaturation
annealing
extension

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24
Q

temp and time for denaturation

A

90-96 * C

20-60secs

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25
Q

temp and time for annealing

A

50-70 *C

20-90 secs

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26
Q

temp and time for extension

A

68-75*C

10-60 secs

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27
Q

target in pcr

A

double stranded DNA

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28
Q

a process wherein we Reduce double stranded molecules to single stranded molecules

A

denaturation

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29
Q

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

A

denaturation

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30
Q

the most critical step in specificity of the pcr

A

annealing

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31
Q

In this step, there are two oligonucleotides, which will prime the
synthesis of DNA and will anneal to the complementary sequence of the
template.

A

annealing

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32
Q

__determines the specificity of the amplification.

A

Primer

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33
Q

The __ dictates the part of the template that would be amplified.

A

primer

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34
Q

the location where will the primer attach during PCR

A

oligonucleotides

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35
Q

what are the 2 types of primers

A

forward and reverse

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36
Q

the type of primer that will hybridize to the complementary strand just below the 5 prime

A

forward primer

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37
Q

the type of primer that hybridizes to the 3 prime of the sequence to be amplified

A

reverse primer

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38
Q

EXTENSION (PRIMER EXTENSION) also called as

A

elongation

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39
Q

This is when the DNA synthesis occurs.

A

EXTENSION

40
Q

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.

A

EXTENSION (PRIMER EXTENSION)

41
Q

bases always attach to which part of the primer

A

3` end

42
Q

In some cases, the
annealing temperature
is close enough to the
extension temperature
that the reaction can
proceed with only 2
temperature changes.

A

Two-Step PCR

43
Q

COMPONENTS OF PCR

A
  1. Primers
  2. DNA template
  3. Deoxyribonucleotide
    Bases
  4. DNA Polymerases
  5. PCR Buffer
  6. Thermal Cyclers
  7. Controls for PCR
44
Q

Critical component of PCR because they determine the
specificity of the PCR

A

primer

45
Q

primer

They are analogous to the _ in blotting and hybridization procedures

A

probes

46
Q

primers are desifgned using genomic sequence available from

A

national center for biological information

47
Q

factors that affect primers

A
  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
Q

the 2 factors that affecr primer is affected by ___

A

the melting temperature of the primer

49
Q

served as the starting point in setting optimal annealing - starting point for optimal binding

A

melting temperature

50
Q

primers (forward) its melting temp can be adjusted in the length of the template and number of guanine and cytosine

t or f

A

true

51
Q

primers

single stranded DNA fragments that how many bases in length

A

20-30 bases in length

52
Q

problems in primer

A

mispriming
primer dimers

53
Q

creation of unintended product caused by primers attaching to wrong area

A

mispriming

54
Q

how to prevent mispriming

A

HOT start PRC

55
Q

PCR products that are double the size if the primer, results from the binding of primer with each other (on the 3` end)

A

primer dimers

56
Q

it connects all bases to each other

A

phosphodiester bond

57
Q

connects the primer to template

A

hydrogen bond

58
Q

Clinical sample sources of dna template

A

all microorganism

59
Q

what kind of sample if we are using hair follicle

A

mitochondrial DNA

60
Q

PCR reagents and conditions, nanogram amounts of genomic DNA are
sufficient for consistent results

true or false

A

true

61
Q

how many data template are we using?

A

❑Routine clinical analysis - 100 ng to 1 μg of
DNA is usually used

62
Q

Lesser amount of data template are required for more
defined template preparations such as
_____ DNA or product from a
previous amplification

A

cloned target

63
Q

best templates

A

good condition
free of contaminating proteins
without nicks or breaks that can stop DN synthesis or cause misincorporation of nucleotide bases

64
Q

templates with high GC content and secondary structure may prove more ____ to optimize for amplification

A

difficult

65
Q

are
the building blocks of DNA

A

Nucleotide triphosphates

66
Q

Deoxyribonucleotide
triphosphates (dNTPs

A
  1. Adenine
  2. Thymine
  3. Guanine
  4. Cytosin
67
Q

In common PCR applications,
the recommended final
concentration of each dNTP is
generally

A

0.2 mM

68
Q

why does the building block of the DNA, nucleotide triphosphate, must be a triphosphate and not a mono or di-

A

diphosphate results from poor manufacturing conditions and mono and diphosphate are contaminated with heavy metals

69
Q

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 ___

A

Taq DNA polymerase

70
Q

DNA polymerase, kary mullis first performed PCR, he used the DNA polymerase isolated from ___

A

E coli

71
Q

first molecule of the year 1985

A

Taq polymerase - enzyme of PCR

72
Q

Taq polymerase is isolated from

A

thermus aquaticus

73
Q

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

A

true

74
Q

an enzyme derived from thermophilic bacterium that Were subsequently exploited for laboratory use

A

Tth polymerase

75
Q

an enzyme derived from thermophilic bacterium that has reverse transcriptase activity so that it can be used in reverse transcriptase PCR OR rt-pcr

A

tth polymerase

76
Q

an enzyme derived polymerase that have proof-roofing functions and can generate products over 30kbp (30, 000 bases in length)

A

vent polymerase

77
Q

Taq polymerase is first isolated in year ___ at ____

A

1969 at yellow stone national park in california

78
Q

modified version of Taq polymerase

A

stoffel fragments

79
Q

stoffel fragments
modified version of taq polymerase that lack amino acid on the __- terminal postion of taq polymerase

A

289th terminal

80
Q

advantage of stoffel fragments

A

has 3 to 5 exonucleases

81
Q

half life of stoffel fragments

A

double in high temp of that of Taq polymerase

82
Q

purpose of stoffel fragments

A

alleles specific PCR and for amplication with high guanine and cytosine contents

83
Q

provides optimal conditions for enzyme activity

A

pcr buffer

84
Q

PCR BUFFR

The buffer pH is usually between ___ and is often stabilized by Tris
HCl.

A

8.0 and 9.5

85
Q

monovalent cations example

A

potassium chloride
ammonium sulfate

86
Q

divalent cation

A

magnesium chloride

87
Q

these affects the annealing and denaturation temperature of DNA and enzyme activity

A

monovalent cation

88
Q

it affects the primer annealing and is very important for enzyme activity

A

divalent cation

89
Q

this will lower the amount of magnesium available for the enzyme

A

EDTA or other chelators

90
Q

Too few Mg2 ions wil cause ___ to enzyme

A

lower enzyme efficiency resulting in a low yield of PCR product

91
Q

overly high Mg2 concentrations will cause ___ to enzymes

A

promote misincorporation and thus increase the yield of nonspecifc products

92
Q

lower Mg2 concentrations will cause ___ to enzymes

A

this concentration is desirable when fidelity of the PCR is critical

93
Q

recommended range of MgCl2 concentration is ____

A

1-4 mM

94
Q

range of MgCl2 in standard reaction conditions

A

1-4 mM

95
Q

Tris buffer concentration ____ and accessory buffer components are also important for optimal enzyme activity and accurate amplification of the intended product

A

10 mM

96
Q

purpose of accessory components

A

used to optimize reactions

97
Q

an accessory component that binds inhibitors and stabilizes the enzyme

A

bovine serum albumin (10-100 ug/ml)

98
Q

an accessory component that provides reducing conditions that may enhance enzyme activity

A

dithiothreitol (0.01mM)

99
Q

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

A

formamide (1-10%)

100
Q
A
101
Q

substitute for dithiothreitol

A

beta 2 mercaptoethanol

102
Q

aside from the accessory components we tackle about, we can add ___ as well to allow polymerase extensions in difficult aea

A

GITC guanidine isothiocyanate
triton X100
glycerol
dimethyl sulfoxide

103
Q
A

MODX (16 decks)

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