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MODX > week7: NUCLEIC ACID ISOLATION ANDTECHNIQUESINEXTRACTION > Flashcards

week7: NUCLEIC ACID ISOLATION ANDTECHNIQUESINEXTRACTION Flashcards

1
Q

a process of reducing a very pure nucleic acid, DNA and RNA, that is free from contaminants

A

extraction of nucleic acid

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2
Q

goal of extraction of nucleic acid

A

to produce a very pure nucleic acid to use in nucleic acid testing such as PCR, blotting nucleic acid, and gel electrophoresis

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3
Q

2 main methods used in extraction

A

precipitation and centrifugation1

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4
Q

1869 the first one who isolated nucleic acid

A

friedrich miescher

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5
Q

friedrich miescher

He discovered it when he was studying the chemical nature of the nuclei of the ___

A

white blood cell

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6
Q

a method that friedrich miescher used to extract nucleic acid

A

precipitation

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7
Q

the key discoverer of semi-conservative model of DNA

A

meselson and stahl

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8
Q

what methods do meselson and stahl used to discover the semi conservicative model of DNA

A

centrifugation - density

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9
Q

Nucleic acid extraction can be divided into three
steps:

A

breaking open tissues and cells
removing proteins/lipids/contaminants
transferring the nucleic acid to water or buffer solution that will preserve them without interfering with subsequent work

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10
Q

breaking open tissues and cells is also called as

A

cell lysis

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11
Q

mechanism to lyse a cell is

A

physical and chemical

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12
Q

example of chemical lysing agent

A

on our experiment, we use of detergent

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13
Q

what is the most commonly used
type of lysis buffer?

A

1-2% sodium dodecyl sulfate

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14
Q

a reagent that will help to remove the protein contaminants

A

proteinase K

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15
Q

a reagent that will help to remove the cell wall of gram negative bacteria

A

lysozymes

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16
Q

a process called as removing contaminants is

A

homogenization or purification

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17
Q

a reagent that will help to remove the peptidoglycan of gram positive bacteria

A

aminopeptidase

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18
Q

the substep under the purification of nucleic acid

A

precipitation using alcohol

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19
Q

why alcohol is used in precipitation of nucleic acid

A

insoluble in alcohol

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20
Q

the purpose of the last step of nucleic acid extraction

A

for solubilization of the nucleic acid

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21
Q

what is the media used in solubilization of the nucleic acid / resuspension

A

eluting buffer
- tris HCL
- tris acetate EDTA buffer
- molecular grade water

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22
Q

the last step of extracting DNA is called is

A

elution

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23
Q

the product of elution is called as

A

eluate

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24
Q

CONSIDERATIONS FOR CHOOSING ISOLATION
METHOD

A
  1. specimen type - cellularity
  2. amount of sample and desired yield
  3. purity and size of isolate
  4. ease of operation and throughout
  5. cost and hazards
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25
Q

the DNA extracted from organism is called as

A

plasmid

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26
Q

3 main types of nucleic acid isolation

A

solid phase isolation
liquid phase isolation
chelex 100 method

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27
Q

the easiest method of isolation

A

solid phase

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28
Q

the most expensive method of isolation

A

chelex 100

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29
Q

the cheapest method of isolation

A

liquid phase isolation

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30
Q

liquid phase isolation uses phenol and chloroform. What are its downside

A

phenol can cause burns and chloroform is carcinogenic

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31
Q

this isolation method is cheap but has low yield of nucleic acid

A

liquid phase isolation

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32
Q

SAMPLES FOR MOLECULAR DIAGNOSTIC

the bacteria used for extraction is what gram type of bacteria

A

gram negative bacteria

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33
Q

SAMPLES FOR MOLECULAR DIAGNOSTIC

the fungi used for extraction is extracted in what part

A

yeast form and in hyphal structure

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34
Q

SAMPLES FOR MOLECULAR DIAGNOSTIC

the virus used for extraction is contaminated by ___

A

the dna of the host since they disintegrate themselves on the host cell

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35
Q

SAMPLES FOR MOLECULAR DIAGNOSTIC

the virus used for extraction is performed by this method ___

A

centrifugation

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36
Q

SAMPLES FOR MOLECULAR DIAGNOSTIC

the worms used for extraction is ___ prior to extraction

A

teased, grinded, minced

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37
Q

SAMPLES FOR MOLECULAR DIAGNOSTIC

the bacteria used for extraction is collected in what phase ___

A

harvested that grows in less than 24 hrs incubated
in mid logarithmic phase which means it’s activately dividing and growing

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38
Q

SAMPLES FOR MOLECULAR DIAGNOSTIC

the blood used for extraction is ___

A

the one that is collected in tripotassium EDTA (EDTA K3) and acid-citrate-dextrose

purple and yellow top

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39
Q

SAMPLES FOR MOLECULAR DIAGNOSTIC

the forensic science we need for extraction is ___

A

chain of custody

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40
Q

why heparin is not used for extraction if using blood as sample

A

2 reason
- coextracted in the DNA extraction
- it inhibits enzymes such as reverse transcriptase and taq polymerase

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41
Q

SAMPLES FOR MOLECULAR DIAGNOSTIC

the tissue for extraction is ___

A

fresh and reserve

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42
Q

SAMPLES FOR MOLECULAR DIAGNOSTIC

the human cell used for extraction is ___

A

hair shaft, cells in nasopharyngeal and oropharyngeal

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43
Q

SAMPLES FOR MOLECULAR DIAGNOSTIC

the stool sample used for extraction is ___ prior extraction

A

digestion and decontamination

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43
Q

SAMPLES FOR MOLECULAR DIAGNOSTIC

the sediments used for extraction is ___ prior the extraction

A

centrifuge

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44
Q

SAMPLES FOR MOLECULAR DIAGNOSTIC

the plant used for extraction is extracted in what area

A

root and stem

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45
Q

MOSTCOMMONLYUSEDDNAEXTRACTION
PROCEDURES

A

liquid, solid, chelex 100

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46
Q

LIQUID PHASE ISOLATION METHOD

what are its 2 types

A

organic and inorganic

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47
Q

organic extraction of liquid-phase isolation method is through

A

phenol and chloroform which can cause burns and carcinogenic

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48
Q

inorganic extraction of liquid-phase isolation method is through the use of

A

proteinase K and salting out

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49
Q

salting out has what concentration of acid and salt

A

low pH and high salt concentratrion

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50
Q

Solid PHASE ISOLATION METHOD

what are its 2 types

A

manual method (silica-based method)
automated method (magnetic beads isolation)

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51
Q

the first type of solid phase extraction that is known as silica-based method uses specialized column with silica is called as

A

spin column

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52
Q

the two type of solid phase isolation (silica based and magnetic beads) follows the same process which are

A

adsorption and elution

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53
Q

the process which the solid phase method follows and is about binding and washing of nucleic acid

A

adsorption

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54
Q

the process which the solid phase method follows and is about transferring of nucleic acid for resuspension

A

elution

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55
Q

most preferred method and most commonly used isolation method

A

solid phase isolation

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56
Q

this type of isolation method is the most sensitive and is expensive

A

chelex 100

57
Q

chelex 100 method uses ____ beads which is used enhance the attachment of polyvalent metal ions leaving the DNA in the solution

A

resin

58
Q

this type of isolation of only small amount of sample or minimal sample

e.g. fungal extraction and forensic studies

A

chelex 100

59
Q

the old method of nucleic acid isolation and is used to develop the semiconservative model of dna and is used for dna isolation

A

DENSITY GRADIENT CENTRIFUGATION STRATEGIES

60
Q

DENSITY GRADIENT CENTRIFUGATION STRATEGIES
it divies the DNA into 2 bands called

A

main band
satellite band

61
Q

DENSITY GRADIENT CENTRIFUGATION STRATEGIES

used ___ to allow the separation of the cells in the sample

A

preformed gradient

62
Q

preformed gradient examples

A

sucrose and cesium chloride

63
Q

the preformed gradient used in isolation of blood

A

sucrose = ficoll

64
Q

preformed gradient initially used for DNA classification

A

cesium chloride

65
Q

the main band in density gradient centrifugation strategies is rich in ___

A

adenine and thymine

66
Q

the satellite band band in density gradient centrifugation strategies is rich in

A

cytosine and guanine

67
Q

In separation of target cells/viruses

viruses are separated thrpugh

A

centrifugation

68
Q

what is the reason why we need to prevent rbc contamination in wbc extraction

A

rbc will be hemolyzed in the process and the hemoglobin will inhibit the extraction of wbc

69
Q

in older method, bacteria are separated from contaminating materials using

A

1% SDS and strong base solution (0.2M Sodium hydroxide NAOH )

70
Q

this solution prevents the
Nucleic acid from enzymatic degradation of hydrolases
(e.g., phosphatases, glycosidases, and proteases)

A

7-9Murea, 2Mthiourea, or 2% SDS

71
Q

disadvantage using 0.2 M NaOH (strong alkaline solution)

A

the nucleic acid will turn single stranded - denatured

72
Q

3 main action of hydrolysis in nucleic acid

A

remove hydrogen bonds in between bases
destroy the phosphodiester bond
attacking the OH group

73
Q

which one is more labile in terms of hydrolasis

rna or dna

A

rna as they have OH

74
Q

The use of a cationic polymer to precipitate nucleic acids in 1M NaCl, where proteins remain in supernatant (PEI must be removed before further analyses)

A

Polyethyleneimine

75
Q

Cell extracts are denatured with high temperatures causing them to precipitate. Stability is enhanced

A

Thermal

76
Q

Polyethylene Glycol Concentration of PEG unique to the protein mixture is added. Due to the excluded volume principle and centrifugation, precipitated proteins are pelleted (PEG must be removed before further analyses)

A
  • Nonionic polymer
77
Q

The choice of lysis buffer depends on the _____in
preparation methods.

A

protein target of extraction, sample size, and experience

78
Q

a lysis buffer that is usually used in
boiling of bacteria

A

Triton X-100

79
Q

Saturation of salt to precipitate protein

A

salting out

80
Q

salting out may be in a form of __

A

ammonium sulfate or sodium sulfate
sodium acetate

81
Q

the main mechanism of salt in salting out

A

removal of histone bounded in the DNA

82
Q

Centrifugation at high speed using molecular weight
cutoff filter to remove contaminants

A

ultracentrifugation

83
Q

isoelectric point uses what component

A

pH

84
Q

a substance that is added in protein mixture that uses nonionic polymer technique

A

polyethylene glycol concentration

85
Q

the preferred sample cell for WBC q

A

mononuclear cells

86
Q

two ways to isolate WBC

A

differential density gradient centrifugation
differentitial lysis

87
Q

In DIFFERENTIAL DENSITY GRADIENT CENTRIFUGATION, what is the component used

A

Ficoll, which is a sucrose

88
Q

is a highly branched sucrose
polymer that does not penetrate biological
membranes.

A

Ficoll

89
Q

It is used in biology laboratories to separate
blood into its components.

A

ficoll

90
Q

Differential lysis is also known as

A

differential
extraction

91
Q

process in which
DNA from two different type of cells can be
extracted without mixing their content.

A

differential extraction

92
Q

this type of extraction method if nucleated cells that take advantage of the osmotic fragility of RBC and WBC

A

Differential lysis

93
Q

Fixed embedded tissue has to be deparaffinized
by soaking in __

A

xylene

94
Q

Wholetissue samples are disrupted by grinding
the frozen tissue in __, homogenizing
the tissue, or simply mincing the tissue using a scalpel.

A

liquid nitrogen

95
Q

the ideal fixative in fixed tissue for dna extraction

A

10% neutral buffered formalin and acetone

96
Q

the fixative to prevent in tissues

A

mercuric based fixative

Bouin’s & B-5 Substitute Fixatives
carnoy’s

97
Q

Lesstoxic xylene substitutes, such as
___, are also often used for this
purpose

A

Histosolve, Anatech Pro-Par, or
ParaClear

98
Q

the main type of extraction for tissue sample is the

A

proteinase K

99
Q

are commercially
available for microorganism for extraction

A

Lysozyme or zymolyase

100
Q

in the extraction of microorganism

Alternatively, cell walls can be broken mechanically.
○ ______

A

By grinding or by vigorously mixing with
glass beads.

101
Q

we are using gentler enzymatic procedure involving lysozyme to extract nucleic acid from microorganism specially if the method we are using is ___

A

restriction fragment length polymorphism (abbreviated RFLP)

102
Q

to remove the polysaccharide membrane in fungi, we are using

A

Cetyl trimethyl ammonium bromide (CTAB)

103
Q

a chelating resin that has high affinity for polyvalent metal ions

A

chelex 100

104
Q

purpose of magnesium and calcium in the extraction

A

are polyvalent metal ions that increases kinase enzyme

105
Q

this isolation method that is for high amount of nucleic acid and is usually for fungal and forensic method

A

chelex 100

106
Q

the use of phenol and chloroform method

A

DNA organic isolation method

107
Q

3 layers of DNA organic isolation method formed

A

upper aqueous layer
interface layer
organic layer

108
Q

the layer formed in dna organic isolation method where the DNA is found

A

upper aqueous layer

109
Q

the layer formed in dna organic isolation method where the all cellular/white debri is found

A

interface layer

110
Q

the layer formed in dna organic isolation method where the all the phenol and chloroform as well as lipid is found

A

organic layer

111
Q

the most important layer in dna organic isolation method

A

upper aqueous layer

112
Q

after getting the upper aqueous layer, what’s next in the step?

A

precipitate the nucleic acid using alcohol

113
Q

rules in aqueous and alcohol ratio

A

isopropanol - 1:1
ethanol 1:2 or twice the amount of ethanol

114
Q

why do we need to increase the amount of ethanol if we’re using such

A

it evaporates fast

115
Q

purpose of the DNA precipitation

A

to form a pellet or a solid clump at the bottom f the microcentrifuge tube

116
Q

how do you make sure that there’s no alcohol left

A

wash in 75% alcohol

117
Q

what is being precipitated in dna inorganic isolation method

A

protein, leaving the nucleic acid in the solution

118
Q

this solid phase isolation that are fast, easy to perform and
economical.

A

silica-based method

119
Q

Silica-based nucleic acid purification methods employ a simple __ process

A

bind-wash-elute

120
Q

in the silica-based method, the __ will bind to the nucleic acid/dna

A

silica

121
Q

in the silica-based method, what will happen to other components other than the dna

A

filtered out

122
Q

usually used in extraction of RNA and is used in silica based method that disrupts macromolecules and DEGRADE all the RNASE present on the solutions

A

chaotropic agent

123
Q

example of chaotropic agent

A

guanidine isothiocyanate (GITC)

124
Q

principle of magnetic bead extraction

A

the positive charge magnet will selectively attract the negatively charge DNA in optimized conditions.

washing to remove the contaminants

wash the magnet to eluate in the solution

125
Q

Is abroad-spectrum serine protease

A

proteinase K

126
Q

__ is commonly used in molecular biology
to digest protein and remove contamination from preparations of nucleic acids.

A

Proteinase K

127
Q

Proteinase K is also stable over a wide pH range of

A

4-12

128
Q

isolation of mitochondrial dna

A

first and second approach

129
Q

the approach in isolation of mitochondrial dna in which the sample must be only a hairshaft

A

second approach

130
Q

in second approach , if we use a sample that is not a hairshaft will get what type of dna

A

total dna = nuclear dna + mtdna

131
Q

to identify which is the mtdna among the total dna = nuclear dna + mtdna

A

pcr and hybridization procedure

132
Q

to remove RNASE as well, aside from GITC, we can use

A

Diethyl pyrocarbonate (DEPC)

133
Q

main purpose is to make water RNASE free

A

Diethyl pyrocarbonate (DEPC)

134
Q

difference of ORGANIC RNA EXTRACTION to DNA organic extraction

A

addition of isoamyl alcohol

135
Q

purpose of addition of isoamyl alcohol

A

remove foaming as it subsequently affect the extraction of RNA

136
Q

how do we extract mRNA and not the total RNA

A

use the isolation of poly A (messenger) RNA

137
Q

this process uses single stranded nucleic acid and has high binding to adenine

A

isolation of poly A (messenger) RNA

138
Q

the short stretch of single stranded nucleic acid called as

A

oligo(dt) probe

139
Q

this short probe can be used to purify mRNA from other RNA

A

oligo(DT ) probes

140
Q

problem in isolation method of poly A (messenger) rna

A

final product is not that purified as no purification method is used

141
Q
A

MODX (17 decks)

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