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MOL. BIO LEC > MIDTERM: DNA ISOLATION > Flashcards

MIDTERM: DNA ISOLATION Flashcards

1
Q
  • Key to the molecular biology
  • Removal of nucleic acids (DNA and/or RNA) from the cells in which they normally reside.
A

Nucleic Acid Extraction

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2
Q

APPLICATIONS OF NUCLEIC ACID EXTRACTION

A

DIAGNOSING DISEASE AND GENETIC DISORDERS
- FORENSICS
- PATERNITY TESTS
- ANCESTRY TRACKING
- GENETIC ENGINEERING

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3
Q

Target Nucleic Acids: free from contamination with macromolecules such as:

A

PROTEINS, CARBOHYDRATES, LIPIDS OR OTHER NUCLEIC ACIDS

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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 (1869)

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5
Q

process where the substance settle at the bottom of tube (the DNA)

A

PRECIPITATION

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5
Q

Developed from density-gradient centrifugation strategies

A

Early Routine Laboratory Procedures of DNA Isolation

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6
Q

demonstrate the semi-conservative replication of DNA

A

 Messelson & Stahl (1958)

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7
Q

daughter DNA consists is consist of old and new strand, preserve the integrity of the parent strand

A

Semiconservative

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8
Q
  • Can be lysed by high pH (alkaline) and detergents
  • Break/lyse the cell wall by adding detergent or high pH
A

A. Bacteria and Fungi

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9
Q

thin cell layer

A

Gram +

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10
Q

thick cell layer of peptidoglycan (

A

Gram -

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11
Q

Some bacteria with tough cell walls:
- uses the enzyme “lysozyme”

A

ENZYMATIC DIGESTION

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12
Q

grinding/vigorously mixing with glass beads
- Grind the thick cell wall, physically break open the bacteria cell)

A

MECHANICAL METHOD

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13
Q

detergent (1% sodium dodecyl sulfate/SDS) and a strong base (0.2M NaOH) in the presence of Tris base, ethylenediaminetetraacetic acid (EDTA; anticoagulant) and glucose.

A

CHEMICAL LYSIS

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14
Q

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

A

YEAST FILAMENTOUS FUNGI & GRAM + BACTERIA

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15
Q

o Held within free viruses or integrated into the host genome along with host DNA.
o Cell-free specimens (plasma) will be used for viral detection
o May require concentration of viroids by centrifugation or other methods

A

Viral DNA

16
Q

naked virus, no protein capsule basic structure of virus

A

VIROIDS

17
Q

o Obtained from the blood plasma
o Purified of red blood cells (RBCs) & other components by either differential density-gradient centrifugation or differential lysis

A

Nucleated Cells in Suspension (Blood & Bone Marrow Aspirates)

18
Q

 Exosomes (release by cancer cells)
o Small vesicles (30 – 100 nm in diameter), which form by imagination & budding from the inside of cellular endosome vesicles and are secreted by living cells
o Contain nucleic acid and can be collected through centrifugation
o Diagnostic & prognostic analyses purposes (liquid biopsy)
o Solid-phase collection of nucleic acid

A

Plasma

19
Q

Dissolved by mincing the tissue

A

FRESH TISSUE

20
Q

Dissociated by grinding and homogenizing the tissue

A

FROZEN TISSUE

21
Q
  • preserve tissue morphology
  • 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 (high contamination if not dewaxed)
  • tissue fixatives affect the quality of DNA
A

FIXED EMBEDDED TISSUE

22
Q

mercury based fixatives that can damage DNA

A

B-5 and BOUIN’S

23
Q

DNA ISOLATION CHEMISTRIES
* uses a combination of HIGH SALT, and 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

24
Q
  • Does not include the organic extraction step
  • Sometimes called “SALTING OUT”, makes use of LOW pH and high salt conditions to selectively precipitate proteins, leaving the DNA in the solution
  • Excess salt is removed by rinsing the pelted nucleic acid in 70% ethanol, centrifuging & discarding the supernatant, then dissolving the DNA pellet in rehydration buffer (10 mM, Tris, ImM EDTA, or water)
A

INORGANIC ISOLATION METHOD

25
Q
  • Used SOLID MATRICES (silica-based products) in the form of columns or beads to bind & hold the DNA for washing
A

C. SOLID PHASE ISOLATION

26
Q

NON – INVASIVE HUMAN DNA ISOLATION

A
  1. GENOMIC DNA PURIFICATION FROM HAIR
  2. GENOMIC DNA PURIFICATION FROM SALIVA
  3. GENOMIC DNA EXTRACTION FROM URINE SAMPLE
27
Q

→ Hair sample is incubated (95°C) for 10 mins in NaOH (strong base/alkali) buffer & the supernatant is subjected to DNA purification after centrifugation

A

Simple Alkaline Lysis Methods (root of hair)

28
Q

→ Hair sample is incubated (56°C) for 2 hours with buffer containing Tris= HCL, EDTA, NaCl, SDS, DTT and proteinase K, followed by gentle mixing and incubation (60 °C) for 2 hours or until the hair dissolved completely and DNA can be extracted from the solution

A

Smooth Chemical Digestion Method using Dithioreitol (DTT)

29
Q

cells found in the saliva, exfoliated buccal epithelial cells and other cells

A

GENOMIC DNA PURIFICATION FROM SALIVA

30
Q

→ Suspended in Lysis buffer which include Tris, EDTA, SDS & proteinase K
→ Incubated (56°C) for 1-3 hours until the tissue is totally dissolved and DNA

A

Buccal Swabs

31
Q

→ Samples from saline rinse need to be processed/frozen immediately after collection
→ To inhibit growth of bacteria
→ Alcohol containing mouthwash must be used (kills microorganism and reduce bacterial growth)

A

Mouthwash Method

32
Q
  • Inverted and swirled in a specimen cup to create a homogenous suspension of cells followed by the centrifugation
  • Supernatant is removed and a dry pellet containing cells is chilled (20°C) for 15 mins. Followed by the addition of Lysis buffer (Tris, EDTA, SDS, Proteinase K)
  • Sample is incubated (56°C) for 2 hours and then DNA is extracted from solution
A

GENOMIC DNA EXTRACTION FROM URINE SAMPLE

33
Q

PROTEOLYTIC LYSIS OF FIXED MATERIAL

A
  • PARAFFIN – EMBEDDED SPECIMENS
  • BEFORE LYSIS
  • REAGENTS FOR CELL LYSIS
34
Q

o Dewaxed w/ XYLENE specimens
o Rehydrated before nucleic acid isolation

A

PARAFFIN – EMBEDDED SPECIMENS

35
Q

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

A

BEFORE LYSIS

36
Q

o Simple screens: detergents (SS or triton)
o PCR amplification: mixture of this buffer and proteinase K (capable of breaking down proteins)

A

REAGENTS FOR CELL LYSIS

MOL. BIO LEC (14 decks)

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