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Molecular Diagnosis > DNA isolation > Flashcards

DNA isolation Flashcards

1
Q

What type of specimens are used to extract DNA?

A 
Blood
Buffy coat
Bone marrow
Solid tissue
Lavage fluids
Bacteria, viruses
Fungi
Organelles, mitochondria
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2
Q

How is bone marrow and peripheral blood prepared for dna extraction?

A

Through density gradient centrifugation, differential osmolysis and use of streptokinase

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3
Q

How is tissue prepared for dna extraction?

A

It is freezed or crushed, minced, enzymatic digestion

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4
Q

How are plants/fungi prepared for dna extraction?

A

They’re homogenized, and vortex used with glass beads

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5
Q

What anticoagulants are used to collect blood sample for dna extraction?

A

EDTA (purple top) or acid citrate dextrose (yellow top)

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6
Q

Why is heparin not used?

A

It can inhibit PCR

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7
Q

What type of sample is preferable for microbe or viral nucleic acid?

A

Liquid serum or plasma with no cells

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8
Q

List the four dna extraction methods?

A

Organic, inorganic, solid phase, and crude methods

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9
Q

What is organic extraction method?

A

It’s extraction methods that uses organic chemicals, phenol, chloroform.

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10
Q

What is inorganic dna extraction methods?

A

It’s extraction method that uses inorganic chemicals, detergents, EDTA, acetic acid, salt (salting out, spooling)

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11
Q

What is solid phase dna extraction method?

A

It’s the extraction method in which DNA is immobilized on a solid support, beads, or columns.

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12
Q

What is crude extraction method?

A

This is the extraction method in which DNA is released from cell lysis, but not purified

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13
Q

What is the first step in organic extraction? What does this step do?

A

Cell lysis. It releases DNA from cells.

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14
Q

What does cell lysis require?

A

Enzymes and detergents

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15
Q

What do enzymes do in cell lysis?

A

Enzymes break down proteins or cell walls of bacteria

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16
Q

What proteins do enzymes break during organic dna extraction?

A

Protease
Proteinase K
Lysozyme

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17
Q

What is the role of detergent in cell lysis? Give examples of these detergents?

A

Detergents disturb cell membranes. SDS, TX-100

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18
Q

What is the second step in organic extraction after cell lysis?

A

Addition of the organic phase

19
Q

What is the organic phase added in organic extraction?

A

PCI : Phenol, chloroform, isoamyl alcohol

20
Q

In what proportion is PCI added in organic extraction?

A

25:24:1

21
Q

What is the role of phenol, chloroform and isoamyl alcohol in organic extraction?

A

Phenol is acidic
Chloroform helps define organic and aqueous phases
Isoamyl alcohol prevents foaming

22
Q

What is done after the addition of the organic phase?

A

It is mixed and centrifuged, aqueous layer is extracted and DNA is soluble at that time

23
Q

In what layer is DNA soluble?

A

Aqueous layer

24
Q

What does solid phase dna isolation consist of?

A

Binding dna to a solid column matrix or bead under high salt conditions

25
Q

What are the most common columns used?

A

Spin column

26
Q

List the steps in the solid phase dna isolation

A

Cell lysis
Adding cells to column, spin
Wash, spin
Add elution buffer and spin

27
Q

In organic extraction, what is done after transferring aqueous phase to a new tube?

A

Adding alcohol and high salt concentration

28
Q

What is the use of alcohol and the Hugh salt concentration in organic extraction?

A

DNA is insoluble in alcohol and the high salt promotes precipitation for dna to turn solid in solution

29
Q

What happens when dna is added to column in solid phase dna isolation?

A

DNA binds and waste flows through

30
Q

What is the wash and spin step for?

A

Removing contaminants from column

31
Q

What happens after elution buffer is added?

A

DNA is realeased from column into clean tube

32
Q

What are the steps for crude dna extraction?

A

Mixing beads with specimen
Lysing cells by boiling
Spin down debris and beads
DNA is in supernatant

33
Q

What are the special considerations for RNA isolation?

A

RNA is dynamic so gene expression can change
RNA is temperature sensitive
RNA is very susceptible to degradation

34
Q

How is RNA preserved?

A

RNA must be kept on ice when in use or frozen -80dC for storage

35
Q

What are RNases?

A

These are enzymes that degrade RNA

36
Q

How is DNA quality and quantity most determined?

A

Using spectrophotometry or fluorometry

37
Q

What is the formula for DNA concentration?

A

A(260nm) * 50 ug/ml * dilution factor

38
Q

How is RNA concentration calculated?

A

A(260nm) * 40 ug/ml * dilution factor

39
Q

What is the maximum absorbance of nucleic acids?

A

260 nm

40
Q

How is the quality for DNA or RNA calculated?

A

A260/A280

41
Q

What is the optimum quality of DNA?

A

1.8 but 1.6 to 2.1 is acceptable

42
Q

What is the optimum quality of RNA?

A

2.0 but 1.7 to 2.3 is acceptable

43
Q

What is the maximum absorbance of proteins?

A

280nm

Molecular Diagnosis (11 decks)

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