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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
Q

Small vesicles (30-100 nm in diameter), which form by invagination & budding from the inside of cellular endosome vesicles & are secreted by living cells

A

Exosomes

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26
Q

▪ Contain nucleic acid & can be collected by centrifugation ▪ Diagnostic & prognostic analyses purposes (liquid biopsy)

A

Exosomes

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27
Q

what are 3 type of tissue samples??

A

Fresh Tissue
Frozen tissue
fixed, embedded tissue

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28
Q

Dissociated by mincing the tissue

A

Fresh Tissue

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29
Q

Dissociated by grinding and homogenizing the tissue

A

frozen tissue

30
Q

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

A

Fixed, embedded tissue

31
Q

what are the 2 fixative that is good to use??

A

10% neutral Buffered formalin
acetone

(2.0-5.0)

32
Q

what are the 4 less desirable fixatives??

A

B-5
Carnoy’s
Zenker’s
Bouin’s

33
Q

what are the 5 not as good to use fixatives???

A

Zamboni’s
Clarke’s
Paraformaldehyde
Metharcan
Formalin alcohol acetic acid

34
Q

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

A

Organic Isolation Methods

35
Q

Ethanol =
Isopropanol =

ratioo??

A

2:1
1:1

36
Q

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

A

Inorganic Isolation Methods

37
Q

Uses solid matrices (silica-based products) in the form of columns or beads to bind & hold the DNA for washing

A

Solid-Phase Isolation

38
Q

what are the 3 NONINVASIVE HUMAN DNA ISOLATION

A

Genomic DNA Purification from Hair
Genomic DNA Purification from Saliva
Genomic DNA Extraction from Urine Sample

39
Q

2 methods belonged in Genomic DNA Purification from Hair

A

a. Simple alkaline lysis method
b. Smooth chemical digestion method using Dithiothreitol (DTT)

40
Q

Hair with root is incubated (95˚C) for 10 mins in NAOH buffer & the supernatant is subjected to DNA purification after centrifugation

A

Simple alkaline lysis method

41
Q

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

A

Smooth chemical digestion method using Dithiothreitol (DTT)

42
Q

what are the 2 methods belonged in Genomic DNA Purification from Saliva

A

a. Buccal swabs (cotton swabs/cytobrushes)
b. Mouthwash method

43
Q

Cells found in the saliva: exfoliated buccal epithelial cells & other cells

A

Genomic DNA Purification from Saliva

44
Q

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

A

Buccal swabs (cotton swabs/cytobrushes)

45
Q

▪ Samples from saline rinses need to be processed/frozen immediately after collection
▪ Alcohol-containing mouthwash must be used

A

b. Mouthwash method

46
Q

▪ 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

A

Genomic DNA Extraction from Urine Sample

47
Q

❖ Dewaxed with xylene/other agents
❖ Rehydrated before nucleic acid isolation

A

Paraffin-embedded specimens

48
Q

Cells may be washed by suspension & centrifugation in saline/other isotonic buffers

A

before lysis

49
Q

❖ Simple screens: detergents (SDS or Triton)
❖ PCR amplification: mixture of Tris buffer & proteinase K

A

Reagents for cell lysis

50
Q

❖ 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

A

Chelex

51
Q

❖ Easy-to-use system for collection, preservation, & longterm storage of nucleic acids at room temperature

A

DNA extraction/storage cards

52
Q

ISOLATION OF MITOCHONDRIAL DNA
Approaches:

A
  1. 1st isolate the mitochondria by centrifugation
  2. Isolate total DNA by hybridization or PCR
53
Q

BEFORE ISOLATION OF RNA

A

▪ Strict precautions to avoid sample RNA degradation by ribonucleases (RNases)
▪ RNases must be eliminated or inactivated

54
Q

diethyl pyrocarbonate (DEPC) added to water & buffers; vanadyl-ribonucleoside complexes; & macaloid clays

A

RNases inibitors

55
Q

▪ Allocate a separate RNase-free (RNF) area of the laboratory

A

BEFORE ISOLATION OF RNA

56
Q

Most abundant (80%90%) RNA in all cells

A

rRNA

57
Q

Next most abundant RNA fraction (2.5% to 5%)

A

mRNA

58
Q

Also present (small amount)

A

tRNA & snRNA

59
Q

what are the 2 chemistries in RNA ISOLATION??

A

Organic isolation method
Solid-phase isolation

60
Q

what are the other methods for RNAL ISOLATION

A

• Proteolytic lysis of fixed materials
• Rapid extraction methods
• Isolation of PolyA (messenger) RNA

61
Q

RNA is stabilized from further metabolism or degradation after collection

A

RNA tests (array analysis/quantitative reverse transcriptase PCR)

62
Q

To stabilized RNA immediately upon draw

A

Specialized collection tubes

63
Q

Lysed by osmosis or separated from WBCs by centrifugation

A

Reticulocytes in blood & bone marrow samples

64
Q

Kept frozen in liquid nitrogen/immersed in buffer that will inactivate intracellular RNases

A

Tissue

65
Q

Isolated by chemical lysis/by grinding in liquid nitrogen

A

Bacterial & fungal RNA

66
Q

Isolated directly from the serum, plasma, culture medium, or other cellfree fluids by means of specially formulated spin columns or beads

A

Viral DNA

67
Q

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

A

Organic Isolation Methods
▪ Cell lysis

68
Q

Acid phenol:chloroform:isoamyl alcohol ______ solution efficiently extracts RNA

A

(25:24:1)

69
Q

▪ 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

A

Solid-Phase Isolation Method

70
Q

▪ 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

A

YIELD OF RNA FROM VARIOUS SPECIMEN SOURCE

71
Q

▪ 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

A

PROTEOLYTIC LYSIS OF FIXED MATERIAL

72
Q

▪ 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

A

ISOLATION OF polya (messenger) rna

MOLECULAR BIOLOGY (9 decks)

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