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Molecular Diagnostics > Nucleic acid isolation & purification > Flashcards

Nucleic acid isolation & purification Flashcards

1
Q

large yield, high quality DNA specimens

A 
blood
bone marrow
fresh tissue
lavage fluids
bacteria, viruses
fungi
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2
Q

low yield/reduced quality DNA specimens

A 
dried blood
saliva
bone, teeth
amniotic fluid
hair follicles, hair shafts
buccal cells
CSF
fixed tissue
feces
soil
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3
Q

specimen collection for bone marrow & peripheral blood

A

acid citrate dextrose (ACD)

liquid K3EDTA

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4
Q

specimen prep for tissue

A

mince + enzymatic digestion

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5
Q

specimen prep for bacteria

A

lyse cells w/ detergent & often by vortexing w/ glass beads

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6
Q

specimen prep for fungi

A

homogenize by vortexing w/ glass beads

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7
Q

organic DNA isolation

A

uses organic chemicals: phenol & chloroform

  1. lysis (NaOH, SDS)
  2. acidification (acetic acid, salt)
  3. extraction (phenol, chloroform)
  4. DNA precipitation (ethanol)
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8
Q

Solid phase DNA isolation

A

DNA is immobilized on a solid support, beads or columns

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9
Q

use of alcohol/salts to precipitate DNA

A

DNA is insoluble in alcohols

salts help salt out DNA to increase precipitation efficiency

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10
Q

always resuspend DNA in what buffer?

A

10mM Tris-EDTA buffer 8.0-9.0 pH

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11
Q

disadvantages of organic DNA isolation

A

time/labor intensive
cannot automate
hazardous chemicals
inefficient

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12
Q

Solid-phase DNA isolation methods

A

in the presence of a chaotropic agent (high [salt solution] that denatures substances by interfering w/ all forms of molecular interactions) nucleic acid binds to silica/glass

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13
Q

AL/ALT buffer

A

sodium dodecyl sulfate (SDS)
binding to silica (chaotropic effects)
cell lysis (detergent effects)
denatures proteins (increases proteinase K activity 1000x)
contains guanidine HCL as chaotropic salt

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14
Q

Proteinase K

A

fungal protease
SDS resistant
works at wide range of thermal temps
stable for years at room temp as long as Ca2+ is present

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15
Q

2 washing steps do what

A

remove any residual salts & proteins that would inhibit downstream enzymatic reactions
washes contain ethanol

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16
Q

buffer to elute DNA

A

cannot use water because DNA will acid hydrolyze in water after several months
buffer is 10 mM Tris-HCl pH 8.0-9.0 pH
DNA is stable in this solution for years at 4C

17
Q

DNA extractioni from formalin fixed tissue for PCR

A
  1. remove paraffin w/ xylene
  2. remove xylene with EtOH
  3. digest ( proteinase K, SDS for 1 hour)
  4. heat 90C for an hour to remove crosslinks
  5. complete purification as normal
18
Q

formalin action on tissues/ DNA

A

formalin crosslinks biomolecules & chops DNA into pieces from 100-250 bps

19
Q

‘decaled’ tissue DNA isolation

A

CANNOT use this tissue for any molecular applications

any tissue containing bone is decalcified in order to section

20
Q

isolation of Bacterial DNA from stool

A

necessary for C. diff detectioni

  1. add detergent lysis solutioni & incubate
  2. add chelator resin (binds PCR inhibitors & DNAses)
  3. remove chelator
  4. continue with DNA purification as normal
21
Q

problem bacteria

A

respiratory agents= fungi or mycobacterium or nocardia
cannot use detergent to lyse
use magnetic glass beans

22
Q

short term storage of DNA

A

leave at room temperature

23
Q

medium term storage of DNA

A

days to year

keep at 4C but must have a buffered TE solution

24
Q

long term storage of DNA

A

freeze at -20C must have DNA in buffered solution

25
Q

methods to assess DNA

A 
gel electrophoroesis w/ known standards
fluorometer
spectrophotometry:
concentration ug/ml
yield: concentration x ml
purity: 260/280 ratio >1.6
26
Q

what tests use isolated RNA

A 
BCR-ABL oncogene in CML
viral detection/quantification: 
HCV
HIV
HTLV etc
27
Q

problem w/ using RNA

A

RNA is VERY unstable & degrades readily

instability is due to ribose backbone of RNA & the presence of RNAses

28
Q

handling cellular RNA for BCR-ABL

A

RNA must be isolated immediately or whole blood placed in extraction buffer & frozen @ -80C
RNAse enzymes wont be active at low temperatures

29
Q

handling viral RNA

A

spin down cells & freeze the plasma that contains the virus
virus RNA is protected from RNAses bc of its capsid

30
Q

RNase elimination /removal

A

separate laboratory area
products need to be certified RNase free etc
can add RNasin

31
Q

organic RNA isolation

A
  1. lysis (guianidine isothiocyanate)
  2. extraction (phenol, chloroform)
  3. precipitation (ethanol)
32
Q

solid phase RNA isolation

A
  1. lysis (guiannidine isothiocyanate)
  2. RNA adsorption (low pH)
  3. wash RNA (supplied buffer)
  4. elute RNA (low salt)
33
Q

types of RNA used for testing

A

mRNA = BCR-ABL
rRNA = bacterial ID
viral RNA = viral ID & viral loads

34
Q

RNA storage

A

RNA is resuspended in WATER not buffer
on ice 1-2 hr
-20 for 1-2 weeks
-80C for 1 year

Molecular Diagnostics (6 decks)

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