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MOLECULAR BIOLOGY > 3. NUCLEIC ACID ISOLATIOON - LEC > Flashcards

3. NUCLEIC ACID ISOLATIOON - LEC Flashcards

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1
Q

Release of nucleic acid from the cell

A

Nucleic acid isolation

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2
Q

The initial release of the cellular material is achieved by

A

breaking the cell wall and nuclear membranes with cell lysis

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3
Q

The target material is _____ nucleic acid isolation

A

purified

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4
Q

First isolated DNA from human cells through alkaline lysis method

A

Miescher 1869

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5
Q

Demonstrate semi-conservative replication of DNA

A

Meselson and Stahl 1958

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6
Q

Procedures used extensively for extraction of 1-50 kb plasmid DNA from bacteria during the early days of recombinant DNA technology

A

Alkaline lysis

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7
Q

Expected DNA yield for blood(1ml, 3.5-10 x 10^6 WBs/mL) and buffy coat (1ml blood)

A

50-200ug

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8
Q

Expected DNA yield for bone marrow (1mL)

A

100-500ug

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9
Q

Expected DNA yield for cultured cells (10^7 cells)

A

30-70ug

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10
Q

Expected DNA yield for solid tissue (1mg)

A

1-10 ug

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11
Q

Expected DNA yield for lavage fluids (10ml)

A

2-250ug

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12
Q

Expected DNA yield for Mitochondria (10-mg tissue, 10 cells)

A

1-10ug

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13
Q

Expected DNA yield for plasmid DNA, bacterial culture (100ml overnight culture)

A

350ug - 1 mg

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14
Q

Expected DNA yield for bacterial culture (0.5mL, 0.7 absorbance units)

A

10-35ug

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15
Q

Expected DNA yield for feces (1mg; bacteria, fungi)

A

2-228 ug

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16
Q

Specimens adequate for analysis without DNA amplification

A

Blood
Buffy coat
Bone marrow
Cultured cells
Solid tissue
Lavage fluids
Mitochondria
Plasmid DNA, bacterial culture
Bacterial culture
Feces

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17
Q

Specimens adequate for analysis with DNA amplification

A

Serum plasma, CSF
Dried blood
Saliva
Buccal cells
Bone, teeth
Hair follicles
Fixed tissue
Feces

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18
Q

Expected DNA yield for Serum, Plasma, CSF (0.5mL)

A

0.3-3 ug

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19
Q

Expected DNA yield for dried blood (0.5-1 cm diameter spot)

A

0.04-0.7 ug

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20
Q

Expected DNA yield for Saliva (1ml)

A

5-15 ug

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21
Q

Expected DNA yield for buccal cells (1mg)

A

1-10 ug

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22
Q

Expected DNA yield for bone, teeth (500mg )

A

30-50 ug

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23
Q

Expected DNA yield for hair follicles

A

0.1-.0.2 ug

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24
Q

Expected DNA yield for fixed tissue (5-10x10 micron sections ; 10mm)

A

6-50 u

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25
Q

Expected DNA yield for feces (animal cells, 1mg)

A

2-100 ug

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26
Q

Blood and bone marrow specimens preferred tube

A

Yellow tube with ACD

Acid citrate dextrose for molecular studies

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27
Q

Other tubes for blood and bone marrow

A

Tripotassium / K3 (purple)

Sodium Heparin (brown) and Lithium Heparin (green) - cytogenetics studies

Non-additive tubes (Red) - cell free from specimen

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28
Q

Sample preparation of bacteria and fungi

A

Enzyme digestion
Alkaline extraction
Mechanical Disruption
Boiling extraction

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29
Q

Proteinase K: digests proteins

Lysozyme digests other cell organelles

gentle procedure

A

Enzymatic digestion

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30
Q

Most common way of preparing bacteria and fungi samples

1% sodium dodecyl sulfate and 0.2 M NaOH

EDTA as chelating reagent

Glucose for destroying the cell wall

A

Alkaline Extraction

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31
Q

not usually used as it may also destroy the nucleic acid

grinding if it is solid
glass beads with vigorous shaking if plasmid

A

Mechanical Disruption

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32
Q

Method used if the sample is treated with lysoenzyme

Diluted sucrose
Triton X-100 detergent
Tris buffer
Edta

A

Boiling extraction

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33
Q

Sample preparation for nucleated cells in suspension

A

Differential density-gradient centrifugation

Differential osmotic fragility of RBCs and WBCs

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34
Q

Whole blood or bone marrow mixed with isotonic saline is overlaid with Ficoll

preferred method since it does not penetrate the cell membrane

A

Differential density-gradient centrifugation

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35
Q

Incubation in hypotonic water will result in the lysis of RBC and WBC

A

Differential osmotic fragility of RBCs and WBCs

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36
Q

released from solid tumors and transplanted organs

A

Exosomes

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37
Q

Sources of circulating nucleic acids

Isolation of cell-free nucleic acid requires procedures to concentrate the target nucleic acid before isolation

use of plasma for the purpose of diagnostic and prognostic analysis

A

Liquid biopsy

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38
Q

Liquid biopsy sources

A

Plasma, CSF, ascites, pleural fluid

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39
Q

Extracellular viruses is detected with

A

Liquid Biopsy

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40
Q

Sample preparation for tissue samples

A

Frozen tissue
Grinding
Mincing

Fixed tissue

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41
Q

least damaging among tested fixatives

A

Neutral buffer formalin

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42
Q

worst fixatives for DNA recovery

A

Mercury based fixatives such as Bouin’s and B5

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43
Q

How many base pairs can be obtained from a fixed tissue

A

100 base pairs

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44
Q

Frozen tissue can be grinded in

A

liquid nitrogen and homogenizing tissue

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45
Q

what do we use for deparaffinization

A

xylene and xylol

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46
Q

DNA isolation method

A
  1. DNA Isolation chemistries
  2. Proteolytic Lysis of Fixed Material
  3. Rapid Extraction Methods
  4. Isolation of Mitochondrial DNA
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47
Q
  1. DNA Isolation chemistries
A

a. Organic Isolation Methods
b. Inorganic Isolation Methods
c. Solid-Phase Isolation

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48
Q

Not used organic isolation method today since they are carcinogenic

A

Phenol and chloroform

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49
Q

Isolation of small amounts of DNA from challenging samples such as fungi can be facilitated by pre-treatment with

A

Cetyltrimethylammonium bromide CTAB

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50
Q

Detergent that will separate the polysaccharide from the DNA (chitin)

A

Cetyltrimethylammonium bromide CTAB

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51
Q

can be added in first step or last step of the procedure (organic isolation method)
- Purpose in the 1st step: to remove the presence of RNA
- Purpose in the last step: to remove residual RNA

A

RNAse

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52
Q

dissolves hydrophobic
contaminants such as lipids and lypoproteins

A

Phenol and Chloroform

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53
Q

Most preferred salt for organic isolation method

A

Sodium acetate and sodium chloride

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54
Q

Alternative salt for organic isolation method

A

Potassium acetate and lithium chloride

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55
Q

Procedure of organic isolation method

A

a. Lysis
b. Acidification
c. Centrifuge
d. Extraction
e. Precipitation
f. The DNA at the bottom still has residual salts
g. Resuspend them in buffer, Tris-EDTA or distilled water .

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56
Q

Organic isolation uses ________ for lysis

A

Sodium hydroxide and SDS

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57
Q

What do we use for acidification

A

Acetic acid and Salt

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58
Q

In what step will we add equal amount of phenol and chloroform

A

Extraction

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59
Q

In extraction we will form 3 layers which are the

A

> Aqueous phase: hydrophilic components

> Ampiphilic phase: both hydrophobic & hydrophilic components

> Organic phase: lipids & hydrophobic organic elements.

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60
Q

What will we use to precipitate the DNA

A

ethanol

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61
Q

This method was developed due to dangers of organic isolation

A

Inorganic isolation

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62
Q

Inorganic isolation main disadvantage

A

Salt precipitates protein and not other contaminants

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63
Q

high salt solution is used (Na acetate, NaCl, potassium acetate, lithium chloride)

Also called as “SALTING OUT”

A

Preparation of Inorganic isolation Method

64
Q

method that uses silica based products

more rapid

A

Solid phase isolation

65
Q

source of silica

A

Diatomaceous earth

66
Q

Commonly used to isolate viral and bacterial DNA from serum,
plasma, or cerebrospinal fluid.

A

Solid phase isolation

67
Q

Preparation of solid phase isolation

A

a. 1st washing adsorption
b. 2nd washing - elution

68
Q

Proteolytic Lysis of fixed materials procedure

A

a. use xylene to remove the wax
b. use 70% ethanol to rehydrate
c. place the sample in a Tris-EDTA buffer
d. add proteinase K to lyse the proteins in the sample

69
Q

Cation-chelating resin

A

chelex

70
Q

A suspension of 10% resin beads is mixed with the specimen,
and the cells are lysed by boiling

A

Rapid extraction method

71
Q

This method is most commonly used in forensics and
purification of DNA

A

Rapid Extraction Method

72
Q

Isolation of Mitochondrial DNA two types

A

a. Centrifugation (2-step centrifugation)
b. Isolation of total DNA

73
Q

speed of the first step of centrifugation in isolating mitochondrial DNA

A

700 to 2600 x g

74
Q

What is the purpose of the first centrifugation step in mitochondrial DNA isolation?

A

Separate cell debris from supernatant containing mitochondria.

75
Q

What is produced after the first low-speed centrifugation?

A

lower layer (cell debris), upper layer (supernatant).

76
Q

What does the second high-speed centrifugation step yield?

A

Mitochondrial pellet and supernatant

77
Q

Why is 1% SDS detergent used in mitochondrial DNA isolation?

A

To lyse mitochondria and remove proteins.

78
Q

What is the role of cold ethanol in mitochondrial DNA isolation?

A

To precipitate mitochondrial DNA.

79
Q

What buffer is used to resuspend mitochondrial DNA after precipitation?

A

Tris-EDTA buffer.

80
Q

What method is used for total DNA isolation during mitochondrial DNA extraction?

A

Chemical extraction or proteolytic lysis

81
Q

Why must RNases be eliminated during RNA isolation?

A

RNases degrade RNA, so they must be eliminated or inactivated.

82
Q

What temperatures are ineffective in deactivating RNases?

A

-20°C, 100°C, and 121°C do not deactivate RNases.

83
Q

What temperature is required to decontaminate glassware for RNA isolation?

A

Glassware must be decontaminated at 400°C for four to six hours.

84
Q

What are the types of RNA found in total RNA?

A

rRNA, mRNA, and tRNA.

85
Q

What are RNase-free (RNF) conditions?

A

Conditions where RNase enzymes are eliminated to prevent RNA degradation.

86
Q

What is the RNA yield from 1 mL of blood?

A

1-10 µg.

87
Q

What is the RNA yield from 1 mL of buffy coat ?

A

5-10 µg.

88
Q

What is the RNA yield from 1 mL of bone marrow?

A

50-200 µg.

89
Q

What is the RNA yield from 10^7 cultured cells?

A

50-150 µg.

90
Q

How should RNA samples be stored to prevent degradation?

A

Frozen in liquid nitrogen or immersed in a buffer that inactivates RNases.

91
Q

What is the RNA yield from 1 mg of buccal cells?

A

1-10 µg.

92
Q

What is the RNA yield from 1mm2 of fixed tissue?

A

0.2-3 µg.

93
Q

What is the RNA yield from 1 mg of solid tissue?

A

0.5-4 ug

94
Q

What is the RNA yield from 0.5ml of bacterial culture?

A

10-100 ug

95
Q

Which sample type is preferred for human RNA testing?

A

Bone marrow.

96
Q

Can viral RNA be isolated from cell-free fluids?

A

Yes, using spin columns or beads.

97
Q

RNA isolation methods

A

Organic Isolation
Solid Phase Isolation
Proteolytic lysis and Fixed material
Isolation of PpolyA (messenger) RNA

98
Q

What is used for RNA organic isolation?

A

Detergent or phenol with high salt (0.2 to 0.5 M NaCl).

99
Q

What are the RNase inhibitors used in RNA isolation?

A

Guanidine isothiocyanate (GITC) and 2-mercaptoethanol.

100
Q

What is the ratio of reagents in organic RNA extraction?

A

25:24:1 (acid phenol:chloroform alcohol).

101
Q

What is the role of chloroform in RNA isolation?

A

Enhances nucleic acid extraction by protein denaturation and phase separation.

102
Q

What is the pH of the organic phase in RNA isolation?

A

pH 4-6 (acidic).

103
Q

What is used to optimize RNA adsorption in solid-phase isolation?

A

Commercial reagents supplied with silica-based columns

104
Q

What type of RNA does the solid matrix in RNA isolation adsorb?

A

Single-stranded RNA.

105
Q

Fixatives used in extraction of RNA

A

10% buffered neutral formullin
Acetone
Zambionis
Clarke’s
Paraformaldehyde
Methacam
Formalin-alcohol
acetic acid
Carmoy’s
Zenkar’s
Boun’s

106
Q

What is the desirability and size range of 10% Buffered Neutral Formalin?

A

Desirability: Good
Size Range: 20-50 kh

107
Q

What is the desirability and size range of Acetone?

A

Desirability: Good
Size Range: 20-50 kh

108
Q

What is the desirability and size range of Zambionis?

A

Desirability: Not as good
Size Range: 02-20 kh

109
Q

What is the desirability and size range of Clarke’s?

A

Desirability: Not as good
Size Range: 08-10 kh

110
Q

What is the desirability and size range of Paraformaldehyde?

A

Desirability: Not as good
Size Range: 02-50 kh

111
Q

Question: What is the desirability and size range of Methacam?

A

Desirability: Not as good
Size Range: 07-15 kh

112
Q

What is the desirability and size range of Formalin-Alcohol?

A

Desirability: Not as good
Size Range: 10-40 kh

113
Q

What is the desirability and size range of Acetic Acid?

A

Desirability: Less desirable
Size Range: 01 kh

114
Q

What is the desirability and size range of Carmoy’s?

A

Desirability: Less desirable
Size Range: 07-15 kh

115
Q

What is the desirability and size range of Zenkar’s?

A

Desirability: Less desirable
Size Range: 07-15 kh

116
Q

What is the desirability and size range of Boun’s?

A

Desirability: Less durable
Size Range: 01 kh

117
Q

Why is polyA RNA important?

A

It is important in gene expression.

118
Q

What information does polyA RNA carry?

A

It carries information from the DNA.

119
Q

Why is polyA RNA used instead of rRNA in isolation?

A

It is used instead of rRNA.

120
Q

How does the isolation process affect the yield of mRNA?

A

Enrich the yield of mRNA.

121
Q

What type of oligomers are used in the isolation of polyA RNA?

A

Uses single-stranded oligomers of thymine or uracil.

122
Q

How does the polyA tail interact with oligomers in the isolation process?

A

PolyA tail binds with polyT or polyU oligomers.

123
Q

How is polyA RNA eluted from the column?

A

PolyA RNA is eluted by washing the column with warmed, low-salt buffer.

124
Q

How much polyA RNA is expected from 1 µg of total RNA?

A

1 µg of total RNA = 30-40 ng of polyA RNA.

125
Q

How does the isolation process reduce degradation of RNA?

A

Lessens degradation by degrading nucleases

126
Q

Measurement of Nucleic Acid- Quality and
Quantity

A
  1. Electrophoresis
  2. Spectrophotometry
  3. Fluorometry
  4. Microfluidics
127
Q

What is the purpose of using dyes in electrophoresis?

A

Uses dyes to visualize the sample preparation.

128
Q

What types of nucleic acids does Ethidium Bromide stain?

A

both DNA and RNA

129
Q

What does SybrGreen I stain?

A

Stains DNA.

130
Q

What does SybrGreen II stain?

A

Stains RNA.

131
Q

What is unique about Silver Stain compared to fluorescence stains, and how does its storage affect staining?

A

Not a fluorescence stain; longer storage turns all staining “red”.

132
Q

Which dyes are used for DNA and RNA, and which is used for visual inspection?

A

Ethidium Bromide and SybrGreen I are used for DNA and RNA, while Silver Stain is used for visual inspection.

133
Q

What does a bright signal indicate in the context of supercoiled plasmid DNA?

A

Bright signal from supercoiled plasmid DNA.

134
Q

Where does high-molecular-weight chromosomal DNA typically appear on the gel?

A

Near top of the gel.

135
Q

What are the two distinct bands of ribosomal RNA observed in RNA electrophoresis?

A

28S rRNA
18S rRNA

136
Q

How is the concentration of a sample determined using fluorescent dyes in electrophoresis?

A

Fluorescent dyes quantified by the fluorescence intensity of the sample aliquot run on the gel; higher intensity = higher concentration.

137
Q

Why is densitometry considered more accurate for measuring band intensity in electrophoresis?

A

More accurate since a standard measure of density is provided.

138
Q

At what wavelength do nucleic acids absorb light?

A

Nucleic acids absorb light at 260 nm.

139
Q

What principle does spectrophotometry follow?

A

Spectrophotometry follows the Beer-Lambert Law.

140
Q

What are the absorptivity constants for double-stranded DNA and RNA?

A

50 = dsDNA
40 = RNA

141
Q

What is the concentration equivalent of one optical density unit at 260 nm for double-stranded DNA and RNA?

A

One optical density unit at 260 nm is equivalent to:
50 mg/L (or 50 µg/mL) of double-stranded DNA
40 µg/mL of RNA

142
Q

How do you calculate the DNA concentration from an absorbance reading of 0.200 and a 1/100 dilution?

A

A DNA preparation diluted 1/100 yields an absorbance reading of 0.200 at 260 nm. Multiply:
0.200 absorbance units × 50 µg/mL per absorbance unit × 100 = 1,000 µg/mL

143
Q

How do you calculate the yield of DNA if the concentration is 1,000 µg/mL and the volume is 0.5 mL?

A

The yield is calculated using the DNA concentration and volume.
1,000 µg/mL × 0.5 mL = 500 µg

144
Q

At what wavelength do organic compounds show peak absorbance?

A

Peak absorbance at 230 nm.

145
Q

At what wavelength does phenol show peak absorbance?

A

Peak absorbance at 270 nm.

146
Q

At what wavelength do proteins show peak absorbance?

A

Peak absorbance at 280 nm.

147
Q

At what wavelength does particulate matter show peak absorbance?

A

Peak absorbance at >330 nm.

148
Q

What does 3,5-diaminobenzoic acid 2HCI (DABA) bind to?

A

Binds to all deoxyribose such as dsDNA & ssDNA.

149
Q

Which base pairs does Hoechst 33258 bind to?

A

Binds to adenine & thymine base pairs.

150
Q

What is the detection limit of DNA using Hoechst 33258

A

Can detect 250 µg/mL of DNA.

151
Q

Used for specifically detecting dna

A

Hoechst 33258

152
Q

What technology is utilized in microfluidics?

A

Uses lab-on-a-chip technology.

153
Q

Where is the sample applied in microfluidics?

A

Sample is applied to a multi-well chip.

154
Q

How does the sample move in a microfluidic system?

A

Sample moves through microchannels across a detector.

155
Q

What configurations can the instrument software generate in microfluidics?

A

Instrument software generates images in electropherogram (peak) or gel (band) configurations.

MOLECULAR BIOLOGY (18 decks)

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