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Molecular Biology > Chapter 3: Nucleic Acid Extraction Methods (DNA) > Flashcards

Chapter 3: Nucleic Acid Extraction Methods (DNA) Flashcards

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1
Q

first isolated DNA from human celss in 1869

A

Miescher

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2
Q

demonstrated semiconservative replication of DNA in 1958

A

Mesehlson and Stahl

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3
Q

early routine laboratory procedures for DNA isolation were developed from

A

density gradient centrifugation strategies

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4
Q

later DNA isolation procedures tooak advantage of

A

solubility differences

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5
Q

5 types of samples for DNA isolation

A
  1. bacteria and fungi
  2. virus
  3. nucleated cells
  4. plasma
  5. tissue
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6
Q

cells walls are not thick and can be lysed by

A

high pH
detergents

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7
Q

cell walls can be broken down by 2 methods

A

enzymatic
machanical

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8
Q

less likely to damage chromosomal DNA

A

enzymatic methods

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9
Q

much preferred for larger chromosomal targets than plasmid DNA

A

enzymatic methods

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10
Q

Alkaline procedure of cell lysis

A

detergent (1% sodium dodecyl sulfate)
strong base (0.2M NaOH)
Tris Base
EDTA
glucose

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11
Q

Bpiling procedure of cell lysis

A

lysozyme treatment
boiling in diulte sucrose
Triton X 100 detergent
Tris buffer
EDTA

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12
Q

DNA extracted by boiling and alkaline methods are ______, yielding ________ DNA thats i not suitable for __________

A

denatured; single stranded; restriction enzyme analysis

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13
Q

commercial reagents for amplification procedures are used for

A

yeast
filamentous fungi
gram positive bacteria

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14
Q

advantage of commercial extraction

A

speed and simplicity

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15
Q

viral DNA can be in the forms of

A

within free virus
integrated into host genome

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16
Q

viral DNA in free viruses are isolated from

A

cell free specimen like plasma

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17
Q

viral DNA integrated in the host genome can be isolated from

A

nucleated cells in suspension

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18
Q

nucleic acid in human blood and bone marrow comes mostly from

A

WBC

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19
Q

which is better for DNA isolation for blood: clotted blood or anticoagulated blood?

A

anticoagulated blood

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20
Q

two methods to purify WBC of RBCs and other blood components

A

differential density gradient
differentual lysis

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21
Q

Differential density gradients centrifugation mixes whole blood with

A

isotonic saline
Ficoll solution

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22
Q

highly branched sucrose polymer that does not penetrate biological membranes

A

Ficoll

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23
Q

layers of the blood after centeifugation with Ficoll

A

plasma
peripheral blood mononuclear cells (PBMCs)
Ficoll
granulocyte (PMNs)
erythrocytes

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24
Q

in differetial lysis of RBCs, whole blood is incubated with ____ and then centrifuged

A

hypotonic buffer or water

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25
Q

solid tumors and transplanted organs release these into the bloodstream

A

cells
exosomes
nucleic acids

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26
Q

small vesicles which form invaginatiojs and budding from the inside of cellular endosome vesicles and secreted by living cells

A

exosomes

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27
Q

detecying sources of circulating nucleic acids for diagnostic and prognostic analyses

A

liquid biopsy

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28
Q

methods of dissociating fresh or frozen tissues

A

grinding in liquid nitrogen
homogenizing
mincing with scalpel

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29
Q

fixed embedded tissues are deparaffinized and rehydrated by

A

xylene
decreasing content of ethanol

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30
Q

True or false: fixed tossue can be used without dewaxing

A

true

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31
Q

How many base pairs of dna target products can be consistently obtained from fixed tissue

A

100 bp or less

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32
Q

Used to yield longer DNA fragments from tissue

A

proteinase K digestion

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33
Q

Best fixatives to for DNA extraction

A

100% buffered neutral formalin
acetone
(2-5 kb)

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34
Q

worst fixatives to use for DNA isolation

A

B5, Bouin (<0.1 kb)
Carnoy, Zenker (0.7 to 1.5)

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35
Q

4 methods of DNA Isolation

A
  1. Organic isolation
  2. Inorganic isolation
  3. Solid phase extraction
  4. Rapid extraction methods
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36
Q

organic isolation is accomplished through these 4 factors

A
  1. high salt
  2. low pH
  3. phenol
  4. chloroform
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37
Q

Isolation of small amounts of DNA from challenging samples like fungi can be facilitated with pretreatment of

A

Cetyltrimethylammonium bromide (separate from polysaccharides)

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38
Q

prevents RNA contamination

A

RNase

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39
Q

General procedure of Organic Isolation

A
  1. Cells in suspension
  2. Lysis (NaOH & SDS)
  3. Acidification (acetic acid and salt)
  4. Extraction (phenol & chloroform)
  5. Precipitation (ethanol)
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40
Q

forms when ohenol and chloroform are added to cell lysate

A

biphasic emulsion

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41
Q

Layers of the emulsion after centrifugation at the proper pH

A

upper hydrophilic phase with DNA
middle amphiphilic white precipitate
lower hydrophobic phase

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42
Q

DNA in the upper hydrophilic phaseis precipitated by

A

ethanol
isopropanol

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43
Q

ratio of ethanol

A

2:1

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44
Q

ratio of isopropyl

A

1:1

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45
Q

salts use in DNA precipitation

A

ammonium
potassium or sodium acetate
lithium or sodium chloride

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46
Q

ETHANOL OR ISOPROPANOL:
denatured

A

ethanol

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47
Q

ETHANOL OR ISOPROPANOL:
undenatured / pure

A

isopropanol

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48
Q

ETHANOL OR ISOPROPANOL:
more volatile

A

ethanol

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49
Q

ETHANOL OR ISOPROPANOL:
less volatile

A

isopropanol

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50
Q

ETHANOL OR ISOPROPANOL:
does not precipitate at RT

A

ethanol

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51
Q

ETHANOL OR ISOPROPANOL:
precipitates at RT and thus reduces coprecipitation of salt

A

isopropanol

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52
Q

ETHANOL OR ISOPROPANOL:
requires more amount due to its volatility

A

ethanol

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53
Q

ETHANOL OR ISOPROPANOL:
requires less amount

A

isopropanol

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54
Q

ETHANOL OR ISOPROPANOL:
viscous at freezer temperature

A

both

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55
Q

ETHANOL OR ISOPROPANOL:
more practical for large volume samples

A

isopropanol

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56
Q

recovery of minimal.amounts of DNA can be optimized by

A

carrier molecules

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57
Q

earlier carrier molecules used to coprecipitate low concentrations of DNA

A

yeast RNA
glycogen

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58
Q

most recently used carrier moleculez

A

yeast RNA
linear polyacrylamide

59
Q

glycogen carrier molecule is derived from

A

mussels - Mytilus edulis

60
Q

commercial glycogen is treated with ____ to avoid mussel DNA contamination

A

DNase

61
Q

added to commercial glycogen to aid in detecting small pellets

A

color indicator

62
Q

how many carrier molecules are added per mL of cisopropanol micture

A

10-20 micrograms

63
Q

DNA precipitate is collected by

A

centeifugation

64
Q

Excess salt in the DNA precipitate is removed by

A

rinsing pelletes with 70% ethanol
centrifuge
discard supernatant
dissolve in rehydration buffer (10mM Tris, 1mM EDTA)

65
Q

inorganic isolation method was developed due to

A

safety concer about phenol being a caustic reagent

66
Q

inorganic DNA extraction is sometimes called

A

salting out

67
Q

inorganic DNA extraction makes use of

A

low pH
high salt conditions

68
Q

what are the general steps in inorganic DNA extraction

A
  1. Cells in suspension
  2. Lysis (Tris, EDTA, SDS)
  3. Protein precipitation (sodium acetate)
  4. DNA precipitation (isopropanol)
69
Q

protects DNA from damage by environmental DNases and in long term storage

A

EDTA (chelating agent)

70
Q

Inhibits enzyme activity in restriction enzyme digestion or PCR

A

EDTA

71
Q

when DNA yield is low, it is better to dissolve it in

A

water

72
Q

most rapid and comparatively effective DNA extraction

A

solid phase isolation

73
Q

shown to effectively bind DNA in high salt conditions

A

silica based products

74
Q

solid mattrices come in the form of

A

columns or beads

75
Q

most often used columns in the clinical laboratory thaybfir inside microcentrifuge tubes

A

spin columns

76
Q

used specifically for plasmid DNA

A

alkaline lysis

77
Q

general steps of solid phase extraction

A
  1. Cells on suspension
  2. Lysis (supplied reagents)
  3. Acidification (supplied reagents)
  4. DNA adsorption (low pH)
  5. Washing (supplied buffer)
  6. Eluting (low salt)
78
Q

prevents irreversible binding of small amount of target DNA

A

carrier DNA or RNA

79
Q

minimum length of carrier molecule to bind the silica membrane

A

200 nts

80
Q

the cell lysate is applied to a column in _______ buffer

A

high salt

81
Q

In SPE, DNA is eluted with

A

water
TE
low salt buffer

82
Q

washing solutions and eluant can be drawn through the column by

A

gravity
vacuum
centrifugation

83
Q

uses a magnetic resin that holds a specific amount of DNA

A

DNA IQ Promega

84
Q

Promega holds ____ amount of DNA

A

100 ng

85
Q

methodology employed for most robotic DNA isolation systems

A

solid phase extraction

86
Q

provide sufficiently clean DNA for amplification

A

Rapid lysis method and DNA storage cards

87
Q

cation chelating resin that can be used for simple extraction of DNA

A

Chelex

88
Q

General method of Rapid extraction

A
  1. 10% Chelex resin bead mixed with specimen
  2. lyse by boiling
  3. centrifuge
  4. DNA in supernatant is cooled
  5. chloroform
89
Q

two ways to isolate mitochondrial DNA

A

centrifugation
PCR or hybridization

90
Q

centrifugation method of mitochondrial dna isolation

A
  1. homogenize sample bu grinding on ice
  2. centrifuge at low speed (700 - 2600 x g)
  3. centrifuge at high speed ( 10,000 - 16,000 x g)
  4. lyse with detergent
  5. treat with proteinase
  6. precipitate with cold ethanol
  7. resuspend in water or buffer
91
Q

mitochondrial DNA isolation throigh PCR or hybridization

A

extract DNA through previous methods and analyze with PCR or hybridization with total DNA background

92
Q

protect mitochondria from dissociating

A
  1. grind with alkaline buffer and reducing substance (beta-mercaptoethanol)
  2. high ionic strength buffer (lyse nuclear membranes selectively)
93
Q

2 ways of mitochondrial DNA extraction

A
  1. centrifugation
  2. PCR or hybridization
94
Q

steps in centrifugation method of mitochondrial DNA extraction

A

homogenize by grinding on ice
low speed centrifuge (700- 2600 x g)
high speed centrifuge(10k- 13k x g)
lyse with detergent
proteinase
precipitate with cold ethanol
resuspend in water or buffer

95
Q

How to protect mitochondrial.membrane from dissociating

A
  1. grind in alaklaine buffer with reducing agent (B- mercaptoethanol)
  2. selective lysis of nuclear membrane with high ionic strength buffet
96
Q

RNase is active to what temperature

A

-20 and below

97
Q

Reagents for RNAse activation

A
  1. Diethyl Pyrocarbonate (amines become carbamic acid, become insoluble)
  2. vanadyl ribonucleic complex (bind active site
  3. macaloid clay (absorb RNase)
98
Q

mRNA constsis of what percent of total RNA

A

2.5 to 5 %

99
Q

organ with abundant RNase

A

pancreas

100
Q

cell lysis agents in RNA extraction

A

detergent/phenol
high salt (0.2 -0.5 M NaCl
RNase inhibitors
guanidine isothiocyanate
2 mercaptoethanol

101
Q

organic isolation method

A

25:24:1
acid phenol, chloroform, isoamyl alcohol

102
Q

prevents foaming

A

isoamyl alcohol

103
Q

denatures protein and promotes phase separation

A

chloroform

104
Q

acidity allows DNA to become neutral staying in the hydrophobic phase

A

acid phenol

105
Q

enhancers in RNA extraction

A

glycogen
yeast transfer RNA

106
Q

solid phase extraction of RNA is similar to DNA except

A
  1. strong denaturing buffer adjusted before lysate application
  2. filter particulate matter
  3. DNase added directly to adsorbed RNA
107
Q

1 million eukaryotic cells or 10-50 ug of tissue yields

A

10 ug of RNA

108
Q

mRNA isolation

A

single stranded poly U or poly T oligomers immobilized in matrix
wash
elute with warm low salt buffer with detergent

109
Q

mRNA yield per 1 ug of total RNA

A

30-40 ng

110
Q

interferences to mRNA extraction

A
  1. intrastrand/interstrand binding
  2. short polyA tail
  3. A-T rich DNA
  4. copurification of rRNA
111
Q

4 methods of nucleic acid quality and quantity measurement

A

electrophoresis
spectrophotometry
fluorometry
microfluidics

112
Q

stains used in electrophoresis

A

EtBr sybgreen I DNA
EtBr sybgreen II RNA
silver stain (rare)

113
Q

good plasmid DNA

A

bright signal, no band indicating nicked plasmid

114
Q

good high MW choromosomal DNA

A

bright band near top of the gel

115
Q

good RNA

A

2 distinct bands of rRNA

116
Q

most accuratemeasurement of quantity in electrophoresis

A

densitometry

117
Q

other quantitative measurements in electrophoresis

A

fluorescent dyes
visual inspection

118
Q

nucleic acids absorb light at _____ through ______

A

260 nm
adenine residues

119
Q

Beer lambert equation

A

A = €bC

120
Q

absorbance at 260 nm is _____ to concentration of nucleic acids

A

directly proportional

121
Q

1 absorbance unit is equal to
_____ mg/ dL of
dsDNA
RNA
ssDNA

A

50
40
33

122
Q

formula of concentration in ug/mL

A

absorbance x dilution factor x absorbance constant

123
Q

formula for yield in ug

A

concentration x volume of eluant

124
Q

common contaminants and their absorbance

A

230 organic compounds
270 phenol
280 proteins (tryptophan and tyrosine)
330 particulaye matter

125
Q

buffer recommended in spectrophotometric reading

A

alkaline buffer

126
Q

NV of A260/A280 of DNA

A

1.6 to 2.0

127
Q

NV of RNA 260/280

A

2.0 -2.3

128
Q

<1.6

A

protein contamination
- column precipitation
- reprecipitate NA
- repeat protein removal

129
Q

> 2.2

A

RNA contamination
- no resolution if it does not interfere with assay

130
Q

intact DNA measurement is required in assays like

A

next generation sequencing

131
Q

fluorescent dyes used before, until now in detection of nuclei, hybridization control and spot analysis

A

3,5-diaminobenzoic acid HCl (DABA)

132
Q

mechanism of DABA

A

combines with ribose

133
Q

combines with A-T base pairs in minor groove

A

Hiechst 33258

134
Q

complication for Hoechst 22358 and how to resolve

A

high or low CG content
use calf thymust DNA std (50%)

135
Q

sensitivity of Hoechst 33258

A

200 ng/mL

136
Q

more sensitive than Hoechst 22358 and does not bind ssDNA or ssRNA

A

PicoGreen

137
Q

binds short pieces of ssDNA and does not fluoresce with dsDNA or RNA

A

Oligreen

138
Q

fluorescent dye that binds to RNA

A

Ribogreen

139
Q

used to bind RNA but can bind dsDNA

A

Sybgreen II RNA gel stain

140
Q

sensitivity of SybGreen II RNA gel stain

A

2 ng/mL

141
Q

Sybgreen II is _____ lower in mRNA than total RNA

A

20-26%

142
Q

True or False: RNA can be detected in the presence of DNA

A

False, not yet possible

143
Q

lab on a chip technology

A

microfluidics

Molecular Biology (12 decks)

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