Chapter 3: Nucleic Acid Extraction Methods (DNA) Flashcards
1
Q
first isolated DNA from human celss in 1869
A
Miescher
2
Q
demonstrated semiconservative replication of DNA in 1958
A
Mesehlson and Stahl
3
Q
early routine laboratory procedures for DNA isolation were developed from
A
density gradient centrifugation strategies
4
Q
later DNA isolation procedures tooak advantage of
A
solubility differences
5
Q
5 types of samples for DNA isolation
A
- bacteria and fungi
- virus
- nucleated cells
- plasma
- tissue
6
Q
cells walls are not thick and can be lysed by
A
high pH
detergents
7
Q
cell walls can be broken down by 2 methods
A
enzymatic
machanical
8
Q
less likely to damage chromosomal DNA
A
enzymatic methods
9
Q
much preferred for larger chromosomal targets than plasmid DNA
A
enzymatic methods
10
Q
Alkaline procedure of cell lysis
A
detergent (1% sodium dodecyl sulfate)
strong base (0.2M NaOH)
Tris Base
EDTA
glucose
11
Q
Bpiling procedure of cell lysis
A
lysozyme treatment
boiling in diulte sucrose
Triton X 100 detergent
Tris buffer
EDTA
12
Q
DNA extracted by boiling and alkaline methods are ______, yielding ________ DNA thats i not suitable for __________
A
denatured; single stranded; restriction enzyme analysis
13
Q
commercial reagents for amplification procedures are used for
A
yeast
filamentous fungi
gram positive bacteria
14
Q
advantage of commercial extraction
A
speed and simplicity
15
Q
viral DNA can be in the forms of
A
within free virus
integrated into host genome
16
Q
viral DNA in free viruses are isolated from
A
cell free specimen like plasma
17
Q
viral DNA integrated in the host genome can be isolated from
A
nucleated cells in suspension
18
Q
nucleic acid in human blood and bone marrow comes mostly from
A
WBC
19
Q
which is better for DNA isolation for blood: clotted blood or anticoagulated blood?
A
anticoagulated blood
20
Q
two methods to purify WBC of RBCs and other blood components
A
differential density gradient
differentual lysis
21
Q
Differential density gradients centrifugation mixes whole blood with
A
isotonic saline
Ficoll solution
22
Q
highly branched sucrose polymer that does not penetrate biological membranes
A
Ficoll
23
Q
layers of the blood after centeifugation with Ficoll
A
plasma
peripheral blood mononuclear cells (PBMCs)
Ficoll
granulocyte (PMNs)
erythrocytes
24
Q
in differetial lysis of RBCs, whole blood is incubated with ____ and then centrifuged
A
hypotonic buffer or water
25
solid tumors and transplanted organs release these into the bloodstream
cells
exosomes
nucleic acids
26
small vesicles which form invaginatiojs and budding from the inside of cellular endosome vesicles and secreted by living cells
exosomes
27
detecying sources of circulating nucleic acids for diagnostic and prognostic analyses
liquid biopsy
28
methods of dissociating fresh or frozen tissues
grinding in liquid nitrogen
homogenizing
mincing with scalpel
29
fixed embedded tissues are deparaffinized and rehydrated by
xylene
decreasing content of ethanol
30
True or false: fixed tossue can be used without dewaxing
true
31
How many base pairs of dna target products can be consistently obtained from fixed tissue
100 bp or less
32
Used to yield longer DNA fragments from tissue
proteinase K digestion
33
Best fixatives to for DNA extraction
100% buffered neutral formalin
acetone
(2-5 kb)
34
worst fixatives to use for DNA isolation
B5, Bouin (<0.1 kb)
Carnoy, Zenker (0.7 to 1.5)
35
4 methods of DNA Isolation
1. Organic isolation
2. Inorganic isolation
3. Solid phase extraction
4. Rapid extraction methods
36
organic isolation is accomplished through these 4 factors
1. high salt
2. low pH
3. phenol
4. chloroform
37
Isolation of small amounts of DNA from challenging samples like fungi can be facilitated with pretreatment of
Cetyltrimethylammonium bromide (separate from polysaccharides)
38
prevents RNA contamination
RNase
39
General procedure of Organic Isolation
1. Cells in suspension
2. Lysis (NaOH & SDS)
3. Acidification (acetic acid and salt)
4. Extraction (phenol & chloroform)
5. Precipitation (ethanol)
40
forms when ohenol and chloroform are added to cell lysate
biphasic emulsion
41
Layers of the emulsion after centrifugation at the proper pH
upper hydrophilic phase with DNA
middle amphiphilic white precipitate
lower hydrophobic phase
42
DNA in the upper hydrophilic phaseis precipitated by
ethanol
isopropanol
43
ratio of ethanol
2:1
44
ratio of isopropyl
1:1
45
salts use in DNA precipitation
ammonium
potassium or sodium acetate
lithium or sodium chloride
46
ETHANOL OR ISOPROPANOL:
denatured
ethanol
47
ETHANOL OR ISOPROPANOL:
undenatured / pure
isopropanol
48
ETHANOL OR ISOPROPANOL:
more volatile
ethanol
49
ETHANOL OR ISOPROPANOL:
less volatile
isopropanol
50
ETHANOL OR ISOPROPANOL:
does not precipitate at RT
ethanol
51
ETHANOL OR ISOPROPANOL:
precipitates at RT and thus reduces coprecipitation of salt
isopropanol
52
ETHANOL OR ISOPROPANOL:
requires more amount due to its volatility
ethanol
53
ETHANOL OR ISOPROPANOL:
requires less amount
isopropanol
54
ETHANOL OR ISOPROPANOL:
viscous at freezer temperature
both
55
ETHANOL OR ISOPROPANOL:
more practical for large volume samples
isopropanol
56
recovery of minimal.amounts of DNA can be optimized by
carrier molecules
57
earlier carrier molecules used to coprecipitate low concentrations of DNA
yeast RNA
glycogen
58
most recently used carrier moleculez
yeast RNA
linear polyacrylamide
59
glycogen carrier molecule is derived from
mussels - Mytilus edulis
60
commercial glycogen is treated with ____ to avoid mussel DNA contamination
DNase
61
added to commercial glycogen to aid in detecting small pellets
color indicator
62
how many carrier molecules are added per mL of cisopropanol micture
10-20 micrograms
63
DNA precipitate is collected by
centeifugation
64
Excess salt in the DNA precipitate is removed by
rinsing pelletes with 70% ethanol
centrifuge
discard supernatant
dissolve in rehydration buffer (10mM Tris, 1mM EDTA)
65
inorganic isolation method was developed due to
safety concer about phenol being a caustic reagent
66
inorganic DNA extraction is sometimes called
salting out
67
inorganic DNA extraction makes use of
low pH
high salt conditions
68
what are the general steps in inorganic DNA extraction
1. Cells in suspension
2. Lysis (Tris, EDTA, SDS)
3. Protein precipitation (sodium acetate)
4. DNA precipitation (isopropanol)
69
protects DNA from damage by environmental DNases and in long term storage
EDTA (chelating agent)
70
Inhibits enzyme activity in restriction enzyme digestion or PCR
EDTA
71
when DNA yield is low, it is better to dissolve it in
water
72
most rapid and comparatively effective DNA extraction
solid phase isolation
73
shown to effectively bind DNA in high salt conditions
silica based products
74
solid mattrices come in the form of
columns or beads
75
most often used columns in the clinical laboratory thaybfir inside microcentrifuge tubes
spin columns
76
used specifically for plasmid DNA
alkaline lysis
77
general steps of solid phase extraction
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
prevents irreversible binding of small amount of target DNA
carrier DNA or RNA
79
minimum length of carrier molecule to bind the silica membrane
200 nts
80
the cell lysate is applied to a column in _______ buffer
high salt
81
In SPE, DNA is eluted with
water
TE
low salt buffer
82
washing solutions and eluant can be drawn through the column by
gravity
vacuum
centrifugation
83
uses a magnetic resin that holds a specific amount of DNA
DNA IQ Promega
84
Promega holds ____ amount of DNA
100 ng
85
methodology employed for most robotic DNA isolation systems
solid phase extraction
86
provide sufficiently clean DNA for amplification
Rapid lysis method and DNA storage cards
87
cation chelating resin that can be used for simple extraction of DNA
Chelex
88
General method of Rapid extraction
1. 10% Chelex resin bead mixed with specimen
2. lyse by boiling
3. centrifuge
4. DNA in supernatant is cooled
5. chloroform
89
two ways to isolate mitochondrial DNA
centrifugation
PCR or hybridization
90
centrifugation method of mitochondrial dna isolation
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
mitochondrial DNA isolation throigh PCR or hybridization
extract DNA through previous methods and analyze with PCR or hybridization with total DNA background
92
protect mitochondria from dissociating
1. grind with alkaline buffer and reducing substance (beta-mercaptoethanol)
2. high ionic strength buffer (lyse nuclear membranes selectively)
93
2 ways of mitochondrial DNA extraction
1. centrifugation
2. PCR or hybridization
94
steps in centrifugation method of mitochondrial DNA extraction
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
How to protect mitochondrial.membrane from dissociating
1. grind in alaklaine buffer with reducing agent (B- mercaptoethanol)
2. selective lysis of nuclear membrane with high ionic strength buffet
96
RNase is active to what temperature
-20 and below
97
Reagents for RNAse activation
1. Diethyl Pyrocarbonate (amines become carbamic acid, become insoluble)
2. vanadyl ribonucleic complex (bind active site
3. macaloid clay (absorb RNase)
98
mRNA constsis of what percent of total RNA
2.5 to 5 %
99
organ with abundant RNase
pancreas
100
cell lysis agents in RNA extraction
detergent/phenol
high salt (0.2 -0.5 M NaCl
RNase inhibitors
guanidine isothiocyanate
2 mercaptoethanol
101
organic isolation method
25:24:1
acid phenol, chloroform, isoamyl alcohol
102
prevents foaming
isoamyl alcohol
103
denatures protein and promotes phase separation
chloroform
104
acidity allows DNA to become neutral staying in the hydrophobic phase
acid phenol
105
enhancers in RNA extraction
glycogen
yeast transfer RNA
106
solid phase extraction of RNA is similar to DNA except
1. strong denaturing buffer adjusted before lysate application
2. filter particulate matter
3. DNase added directly to adsorbed RNA
107
1 million eukaryotic cells or 10-50 ug of tissue yields
10 ug of RNA
108
mRNA isolation
single stranded poly U or poly T oligomers immobilized in matrix
wash
elute with warm low salt buffer with detergent
109
mRNA yield per 1 ug of total RNA
30-40 ng
110
interferences to mRNA extraction
1. intrastrand/interstrand binding
2. short polyA tail
3. A-T rich DNA
4. copurification of rRNA
111
4 methods of nucleic acid quality and quantity measurement
electrophoresis
spectrophotometry
fluorometry
microfluidics
112
stains used in electrophoresis
EtBr sybgreen I DNA
EtBr sybgreen II RNA
silver stain (rare)
113
good plasmid DNA
bright signal, no band indicating nicked plasmid
114
good high MW choromosomal DNA
bright band near top of the gel
115
good RNA
2 distinct bands of rRNA
116
most accuratemeasurement of quantity in electrophoresis
densitometry
117
other quantitative measurements in electrophoresis
fluorescent dyes
visual inspection
118
nucleic acids absorb light at _____ through ______
260 nm
adenine residues
119
Beer lambert equation
A = €bC
120
absorbance at 260 nm is _____ to concentration of nucleic acids
directly proportional
121
1 absorbance unit is equal to
_____ mg/ dL of
dsDNA
RNA
ssDNA
50
40
33
122
formula of concentration in ug/mL
absorbance x dilution factor x absorbance constant
123
formula for yield in ug
concentration x volume of eluant
124
common contaminants and their absorbance
230 organic compounds
270 phenol
280 proteins (tryptophan and tyrosine)
330 particulaye matter
125
buffer recommended in spectrophotometric reading
alkaline buffer
126
NV of A260/A280 of DNA
1.6 to 2.0
127
NV of RNA 260/280
2.0 -2.3
128
<1.6
protein contamination
- column precipitation
- reprecipitate NA
- repeat protein removal
129
>2.2
RNA contamination
- no resolution if it does not interfere with assay
130
intact DNA measurement is required in assays like
next generation sequencing
131
fluorescent dyes used before, until now in detection of nuclei, hybridization control and spot analysis
3,5-diaminobenzoic acid HCl (DABA)
132
mechanism of DABA
combines with ribose
133
combines with A-T base pairs in minor groove
Hiechst 33258
134
complication for Hoechst 22358 and how to resolve
high or low CG content
use calf thymust DNA std (50%)
135
sensitivity of Hoechst 33258
200 ng/mL
136
more sensitive than Hoechst 22358 and does not bind ssDNA or ssRNA
PicoGreen
137
binds short pieces of ssDNA and does not fluoresce with dsDNA or RNA
Oligreen
138
fluorescent dye that binds to RNA
Ribogreen
139
used to bind RNA but can bind dsDNA
Sybgreen II RNA gel stain
140
sensitivity of SybGreen II RNA gel stain
2 ng/mL
141
Sybgreen II is _____ lower in mRNA than total RNA
20-26%
142
True or False: RNA can be detected in the presence of DNA
False, not yet possible
143
lab on a chip technology
microfluidics
