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MOLECULAR BIOLOGY > MIDTERM LEC: DNA & RNA ISOLATION > Flashcards

MIDTERM LEC: DNA & RNA ISOLATION Flashcards

1
Q

Removal of nucleic acids (DNA and/or RNA) from the cells in which they normally resides

A

NUCLEIC ACID EXTRACTION

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2
Q

PURPOSE IS TO RELEASE NUCLEIC ACID FROM THE CELL FOR:

A
  1. Detecting a specific pathogen (bacteria and viruses)
  2. Diagnosing disease & genetic disorders
  3. Other applications: forensics, paternity tests, ancestry tracking, genetic engineering (vaccines, hormones), etc.
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3
Q

Free from contamination with macromolecules (proteins, carbohydrates, lipids, or other nucleic acids)

A

TARGET NUCLEIC ACID

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4
Q

Isolated the nuclein (DNA) from the WBCs he obtained from the pus on collected surgical bandages in a nearby hospital

A

FRIEDRICH MIESCHER in 1869

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5
Q

demonstrate the semiconservative replication of DNA

A

Meselson & Stahl (1958):

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6
Q

Developed from densitygradient centrifugation strategies

A

EARLY ROUTINE LABORATORY PROCEDURES OF DNA ISOLATION

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7
Q

Solubility differences among chromosomal DNA, plasmids, and proteins in alkaline buffers

A

LATER ROUTINE LABORATORY PROCEDURES OF DNA ISOLATION

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8
Q

Preparing the Sample for DNA ISOLATION

A

• Bacteria & fungi • Viruses • Nucleated cells in suspension (blood & bone marrow aspirates)
• Plasma • Tissue samples

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9
Q

Chemistries in DNA ISOLATION

A

• Organic isolation method
• Inorganic isolation method
• Solid-phase isolation

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10
Q

other chemistries for DNA ISOLATION

A

• Proteolytic lysis of fixed materials
• Rapid extraction methods
• Isolation of mitochondrial DNA (mtDNA)

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11
Q

sample with biggest yield

A

plasmid dna (300 ug-1g)

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12
Q

sample with smallest yield

A

hair follicles (0.1-0.2 ug)

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13
Q

Can be lysed by high pH and detergents

A

Gram-negative bacteria

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14
Q

Some bacteria & fungi with tough cell walls. what method is used???

A

• Enzymatic digestion
• Mechanical method
• Chemical lysis

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15
Q

Commercial reagents designed for isolation of DNA in amplification procedures (PCR)

A

Yeast, filamentous fungi, & gram-positive bacteria:

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16
Q

(lysozyme)

A

Enzymatic digestion

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17
Q

grinding/vigorously mixing with glass beads

A

Mechanical method

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18
Q

detergent (1% sodium dodecyl sulfate) & strong base (0.2 M NaOH) in the presence of Tris base, ethylenediaminetetraacetic acid (EDTA), & glucose

A

Chemical lysis

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19
Q

Held within free viruses/ integrated into the host genome along with host DNA

A

Viral DNA

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20
Q

Cell-free specimens (plasma) will be used for viral detection

A

Viral DNA

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21
Q

May require concentration of viroids by centrifugation or other methods

A

Viral DNA

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22
Q

what are the Nucleated Cells in Suspension???

A

Blood & Bone Marrow Aspirates

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23
Q

Anticoagulants (EDTA/citrate) will be added
▪ Obtained from the blood plasma

A

WBCS

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24
Q

▪ Purified of RBCs & other components by either differential density-gradient centrifugation or differential lysis
▪ Differential osmotic fragility of RBCs & WBCs

A

WBCS

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25
Small vesicles (30-100 nm in diameter), which form by invagination & budding from the inside of cellular endosome vesicles & are secreted by living cells
Exosomes
26
▪ Contain nucleic acid & can be collected by centrifugation ▪ Diagnostic & prognostic analyses purposes (liquid biopsy)
Exosomes
27
what are 3 type of tissue samples??
Fresh Tissue Frozen tissue fixed, embedded tissue
28
Dissociated by mincing the tissue
Fresh Tissue
29
Dissociated by grinding and homogenizing the tissue
frozen tissue
30
May be deparaffinized by soaking in xylene and tissue is rehydrated by soaking it in decreasing concentrations of ethanol • May be used directly without dewaxing
Fixed, embedded tissue
31
what are the 2 fixative that is good to use??
10% neutral Buffered formalin acetone (2.0-5.0)
32
what are the 4 less desirable fixatives??
B-5 Carnoy's Zenker's Bouin's
33
what are the 5 not as good to use fixatives???
Zamboni's Clarke's Paraformaldehyde Metharcan Formalin alcohol acetic acid
34
Use a combination of high salt, low pH, & an organic mixture of phenol & chloroform ❖ Readily dissolves hydrophobic contaminants (lipids & lipoproteins) ❖ Collect cell debris & strips away most DNA-associated proteins
Organic Isolation Methods
35
Ethanol = Isopropanol = ratioo??
2:1 1:1
36
Does not include the organic extraction step ▪ Sometimes called “salting out”, makes use of low-pH & high-salt conditions to selectively precipitate proteins, leaving the DNA in solution
Inorganic Isolation Methods
37
Uses solid matrices (silica-based products) in the form of columns or beads to bind & hold the DNA for washing
Solid-Phase Isolation
38
what are the 3 NONINVASIVE HUMAN DNA ISOLATION
Genomic DNA Purification from Hair Genomic DNA Purification from Saliva Genomic DNA Extraction from Urine Sample
39
2 methods belonged in Genomic DNA Purification from Hair
a. Simple alkaline lysis method b. Smooth chemical digestion method using Dithiothreitol (DTT)
40
Hair with root is incubated (95˚C) for 10 mins in NAOH buffer & the supernatant is subjected to DNA purification after centrifugation
Simple alkaline lysis method
41
Hair sample is incubated (56˚C) for 2h with buffer containing Tris-HCl, EDTA, NaCl, SDS, DTT, & proteinase K, followed by gentle mixing & incubation (60˚C) for 2h or until the hair has dissolved completely & DNA can be extracted from the solution
Smooth chemical digestion method using Dithiothreitol (DTT)
42
what are the 2 methods belonged in Genomic DNA Purification from Saliva
a. Buccal swabs (cotton swabs/cytobrushes) b. Mouthwash method
43
Cells found in the saliva: exfoliated buccal epithelial cells & other cells
Genomic DNA Purification from Saliva
44
Suspended in lysis buffer which includes Tris, EDTA, SDS, & proteinase K ▪ Incubated (56˚C) for 1-3h until the tissue is totally dissolved & DNA is extracted from the solution
Buccal swabs (cotton swabs/cytobrushes)
45
▪ Samples from saline rinses need to be processed/frozen immediately after collection ▪ Alcohol-containing mouthwash must be used
b. Mouthwash method
46
▪ Inverted & swirled in a specimen cup to create a homogenous suspension of cells followed by the centrifugation ▪ Supernatant is removed & a dry pellet containing cells is chilled (-20˚C) for 15 mins followed by addition of lysis buffer (Tris, EDTA, SDS, & proteinase K) ▪ Sample is incubated (56˚C) for 2h & then DNA is extracted from the solution
Genomic DNA Extraction from Urine Sample
47
❖ Dewaxed with xylene/other agents ❖ Rehydrated before nucleic acid isolation
Paraffin-embedded specimens
48
Cells may be washed by suspension & centrifugation in saline/other isotonic buffers
before lysis
49
❖ Simple screens: detergents (SDS or Triton) ❖ PCR amplification: mixture of Tris buffer & proteinase K
Reagents for cell lysis
50
❖ Cation-chelating resin that can be used for simple extraction of DNA from minimal samples ❖ 10% Chelex resin beads is mixed with the specimen & the cells are lysed by boiling
Chelex
51
❖ Easy-to-use system for collection, preservation, & longterm storage of nucleic acids at room temperature
DNA extraction/storage cards
52
ISOLATION OF MITOCHONDRIAL DNA Approaches:
1. 1st isolate the mitochondria by centrifugation 2. Isolate total DNA by hybridization or PCR
53
BEFORE ISOLATION OF RNA
▪ Strict precautions to avoid sample RNA degradation by ribonucleases (RNases) ▪ RNases must be eliminated or inactivated
54
diethyl pyrocarbonate (DEPC) added to water & buffers; vanadyl-ribonucleoside complexes; & macaloid clays
RNases inibitors
55
▪ Allocate a separate RNase-free (RNF) area of the laboratory
BEFORE ISOLATION OF RNA
56
Most abundant (80%90%) RNA in all cells
rRNA
57
Next most abundant RNA fraction (2.5% to 5%)
mRNA
58
Also present (small amount)
tRNA & snRNA
59
what are the 2 chemistries in RNA ISOLATION??
Organic isolation method Solid-phase isolation
60
what are the other methods for RNAL ISOLATION
• Proteolytic lysis of fixed materials • Rapid extraction methods • Isolation of PolyA (messenger) RNA
61
RNA is stabilized from further metabolism or degradation after collection
RNA tests (array analysis/quantitative reverse transcriptase PCR)
62
To stabilized RNA immediately upon draw
Specialized collection tubes
63
Lysed by osmosis or separated from WBCs by centrifugation
Reticulocytes in blood & bone marrow samples
64
Kept frozen in liquid nitrogen/immersed in buffer that will inactivate intracellular RNases
Tissue
65
Isolated by chemical lysis/by grinding in liquid nitrogen
Bacterial & fungal RNA
66
Isolated directly from the serum, plasma, culture medium, or other cellfree fluids by means of specially formulated spin columns or beads
Viral DNA
67
Detergent/phenol in the presence of high salt (0.2 to 0.5 M NaCl) or RNase inhibitors ❖ Guanidine isothiocyanate (GITC) can also be used ❖ Strong reducing agents (2mercaptoethanol) may also be added ❖ DNase is sometimes added
Organic Isolation Methods ▪ Cell lysis
68
Acid phenol:chloroform:isoamyl alcohol ______ solution efficiently extracts RNA
(25:24:1)
69
▪ Begins with similar steps as described for organic extraction ▪ The strong denaturing buffer conditions must be adjusted before application of the lysate to the column & sometimes, ethanol is added
Solid-Phase Isolation Method
70
▪ 1 million eukaryotic cells or 10-50 mg of tissue = 10 μg of RNA ▪ Yield of RNA from biological fluids will depend on the concentration of microorganisms/other target molecules present in the specimen
YIELD OF RNA FROM VARIOUS SPECIMEN SOURCE
71
▪ The quality of RNA from fixed, paraffinembedded tissue will depend on the fixation process & handling of the specimen before fixation ▪ Commercial reagent sets are available for extraction of RNA from fixed-tissue specimens ▪ Specialized reagents / spin columns for removal of genomic DNA contamination are included in some systems ▪ Automated isolation systems have reagent kits & cartridges
PROTEOLYTIC LYSIS OF FIXED MATERIAL
72
▪ RNA often required for analysis: mRNA (2.5%-5% of the total RNA yield) ▪ SS oligomers of thymine/uracil immobilized on a matrix resin column or beads are used ❖ Approximately 30-40 ng of mRNA can be obtained from 1 μg of total RNA
ISOLATION OF polya (messenger) rna

MOLECULAR BIOLOGY (9 decks)

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