Nucleic acid isolation & purification

Question 1

large yield, high quality DNA specimens

Answer 1

blood
bone marrow
fresh tissue
lavage fluids
bacteria, viruses
fungi

Question 2

low yield/reduced quality DNA specimens

Answer 2

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

Question 3

specimen collection for bone marrow & peripheral blood

Answer 3

acid citrate dextrose (ACD)

liquid K3EDTA

Question 4

specimen prep for tissue

Answer 4

mince + enzymatic digestion

Question 5

specimen prep for bacteria

Answer 5

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

Question 6

specimen prep for fungi

Answer 6

homogenize by vortexing w/ glass beads

Question 7

organic DNA isolation

Answer 7

uses organic chemicals: phenol & chloroform

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

Question 8

Solid phase DNA isolation

Answer 8

DNA is immobilized on a solid support, beads or columns

Question 9

use of alcohol/salts to precipitate DNA

Answer 9

DNA is insoluble in alcohols

salts help salt out DNA to increase precipitation efficiency

Question 10

always resuspend DNA in what buffer?

Answer 10

10mM Tris-EDTA buffer 8.0-9.0 pH

Question 11

disadvantages of organic DNA isolation

Answer 11

time/labor intensive
cannot automate
hazardous chemicals
inefficient

Question 12

Solid-phase DNA isolation methods

Answer 12

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

Question 13

AL/ALT buffer

Answer 13

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

Question 14

Proteinase K

Answer 14

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

Question 15

2 washing steps do what

Answer 15

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

Question 16

buffer to elute DNA

Answer 16

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

Question 17

DNA extractioni from formalin fixed tissue for PCR

Answer 17

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

Question 18

formalin action on tissues/ DNA

Answer 18

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

Question 19

‘decaled’ tissue DNA isolation

Answer 19

CANNOT use this tissue for any molecular applications

any tissue containing bone is decalcified in order to section

Question 20

isolation of Bacterial DNA from stool

Answer 20

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

Question 21

problem bacteria

Answer 21

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

Question 22

short term storage of DNA

Answer 22

leave at room temperature

Question 23

medium term storage of DNA

Answer 23

days to year

keep at 4C but must have a buffered TE solution

Question 24

long term storage of DNA

Answer 24

freeze at -20C must have DNA in buffered solution

Question 25

methods to assess DNA

Answer 25

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

Question 26

what tests use isolated RNA

Answer 26

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

Question 27

problem w/ using RNA

Answer 27

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

Question 28

handling cellular RNA for BCR-ABL

Answer 28

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

Question 29

handling viral RNA

Answer 29

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

Question 30

RNase elimination /removal

Answer 30

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

Question 31

organic RNA isolation

Answer 31

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

Question 32

solid phase RNA isolation

Answer 32

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

Question 33

types of RNA used for testing

Answer 33

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

Question 34

RNA storage

Answer 34

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