Nucleic acid isolation & purification Flashcards Preview

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Flashcards in Nucleic acid isolation & purification Deck (34)
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1

large yield, high quality DNA specimens

blood
bone marrow
fresh tissue
lavage fluids
bacteria, viruses
fungi

2

low yield/reduced quality DNA specimens

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

3

specimen collection for bone marrow & peripheral blood

acid citrate dextrose (ACD)
liquid K3EDTA

4

specimen prep for tissue

mince + enzymatic digestion

5

specimen prep for bacteria

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

6

specimen prep for fungi

homogenize by vortexing w/ glass beads

7

organic DNA isolation

uses organic chemicals: phenol & chloroform
1. lysis (NaOH, SDS)
2. acidification (acetic acid, salt)
3. extraction (phenol, chloroform)
4. DNA precipitation (ethanol)

8

Solid phase DNA isolation

DNA is immobilized on a solid support, beads or columns

9

use of alcohol/salts to precipitate DNA

DNA is insoluble in alcohols
salts help salt out DNA to increase precipitation efficiency

10

always resuspend DNA in what buffer?

10mM Tris-EDTA buffer 8.0-9.0 pH

11

disadvantages of organic DNA isolation

time/labor intensive
cannot automate
hazardous chemicals
inefficient

12

Solid-phase DNA isolation methods

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

13

AL/ALT buffer

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

14

Proteinase K

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

15

2 washing steps do what

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

16

buffer to elute DNA

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

DNA extractioni from formalin fixed tissue for PCR

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

formalin action on tissues/ DNA

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

19

'decaled' tissue DNA isolation

CANNOT use this tissue for any molecular applications
any tissue containing bone is decalcified in order to section

20

isolation of Bacterial DNA from stool

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

problem bacteria

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

22

short term storage of DNA

leave at room temperature

23

medium term storage of DNA

days to year
keep at 4C but must have a buffered TE solution

24

long term storage of DNA

freeze at -20C must have DNA in buffered solution

25

methods to assess DNA

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

26

what tests use isolated RNA

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

27

problem w/ using RNA

RNA is VERY unstable & degrades readily
instability is due to ribose backbone of RNA & the presence of RNAses

28

handling cellular RNA for BCR-ABL

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

29

handling viral RNA

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

30

RNase elimination /removal

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

31

organic RNA isolation

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

32

solid phase RNA isolation

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

33

types of RNA used for testing

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

34

RNA storage

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