V L7: DNA isolation and quantification

Question 1

what are the Principle stages of DNA extraction from cells?

Answer 1

• Cell lysis to disrupt cell and nuclear membranes including denaturing and degrading proteins which are abundant in cells and bound to DNA in chromosomes
• Separating DNA from other cell components including cell debris, proteins, lipids, CHO and RNA
• Isolation of the DNA into a aqueous phase.
• Storing DNA for future analyses.

Question 2

what does cell lysis involve?

Answer 2

heating the sample

Question 3

why do we use ethanol precipitation?

Answer 3

because if the DNA is in water it forms a pallet of DNA

Question 4

a detergent does what?

Answer 4

denatures

Question 5

in what ways can we separate and isolate DNA form other cell components?

Answer 5

– Organic or phenol-chloroform method
– Ethanol precipitation
– Silica based extraction (affinity binding) and magnetic separation
– Filtration and centrifugation

Question 6

what does SDS stand for?

Answer 6

sodium dodecyl sulphate

Question 7

what is SDS?

Answer 7

anionic detergent that

denatures proteins.

Question 8

what is proteinase K?

Answer 8

protease (enzyme) that degrades proteins – cleaves peptide bonds.

Question 9

what does EDTA stand for?

Answer 9

Ethylenediamine tetra acetic acid

Question 10

what does EDTA do?

Answer 10

chelating agent, DNase inhibition.

Question 11

what does DTT stand for?

Answer 11

dithiotreitol

Question 12

what is DTT?

Answer 12

reducing agent for disulphide bonds.

Question 13

what does DNase do?

Answer 13

chew up DNA

Question 14

for DNA storage what temperature is needed?

Answer 14

-20 degrees and lower

Question 15

what tube material is best to store DNA in?

Answer 15

PTFE

Question 16

To store DNA we need maintain ____ ____.

Answer 16

pH levels

Question 17

whats an FTA card?

Answer 17

a card containing different chemicals so they can be stored at room temperature

Question 18

what the organic method for storing DNA?

Answer 18

phenol-chloroform method

Question 19

phenol-chloroform method is not used ______.

Answer 19

frequently

Question 20

phenol-chloroform method is used more often on a ___ sample.

Answer 20

dirty

Question 21

phenol-chloroform method involves how many steps ?

Answer 21

2

Question 22

phenol-chloroform method is like a ____ _____ extraction.

Answer 22

liquid - liquid

Question 23

phenol-chloroform method requires the constant removal of a _____ layer.

Answer 23

aquaous

Question 24

phenol-chloroform method involves what 3 steps?

Answer 24

lysis, solvent extraction and DNA seperation

Question 25

the Chelex method involves how many tubes?

Answer 25

1

Question 26

How does the Chelex method work?

Answer 26

1. add chelex resin which inhibits DNAase 2. add proteinase K +boil to denture and degrade 3. centrifuge, pellet at bottom to throw away.

Question 27

what exactly does DNase break?

Answer 27

phosphodiester bonds found in DNA

Question 28

How do chelating agents inactive DNase?

Answer 28

Chelating agents bind with metal ions that are cofactors for DNase, inactivating them.

Question 29

DNA binding method

Answer 29

1. cell lysis + denature proteins, detergent, proteinase K and chaotropic salt. 2. DNA binds to magnetic beads everything else passess through 3. beads collected by magnetic force

Question 30

what are FTA cards sued for?

Answer 30

to store blood and saliva samples.

Question 31

what do FTA cards contain?

Answer 31

- Contains chemicals that causes cell lysis - Protects DNA from nuclease degradation - Prohibits microbial growth

Question 32

vaginal swabs, how to pull out male DNA?

Answer 32

differential lysis

Question 33

How is differential lysis carried out?

Answer 33

SDS EDTA proteinase K, incubate, centrifuge, remove supernatant, spermatozoa are still in tact, add SDS EDTA proteinase K + DTT, centrifuge, disulphide bonds broken, opens membranes of spermatozoa, remove supernatant (male DNA).

Question 34

whats an advantage of bones and hard tissue?

Answer 34

- DNA is protected from degradation | - bone surface can be cleaned with abrasives and bleach or cross-linked with UV

Question 35

what are problems with bones?

Answer 35

- need to be grinded up, hard to obtain homogenous sample | - high PCR inhibitor, high calcium (EDTA can remove it)

Question 36

what can you use to quantify DNA?

Answer 36

Ultraviolet spectroscopy | fluorescent dye

Question 37

Ultraviolet spectroscopy gives you the concentration of what?

Answer 37

DNA protein CHO

Question 38

whats an issue with Ultraviolet spectroscopy ?

Answer 38

it can't differentiate between different types of DNA.

Question 39

Fluorescent dye | _____ to DNA.

Answer 39

binds

Question 40

what two dyes are mainly used?

Answer 40

– Sybergreen | – Ethidium Bromide

Question 41

whats more sensitive Interchelating Fluorescent Dyes or Ultraviolet spectroscopy?

Answer 41

Interchelating Fluorescent Dyes

Question 42

Fluorescence is recorded using what?

Answer 42

spectrofluorometer, or gels

Question 43

Interchelating Fluorescent Dyes requires a...

Answer 43

calibration graph

Question 44

what is the hybridisation method?

Answer 44

- Sample is denatured and a small spot of sample is immobilised onto a nylon membrane - Addition of a 40-nucleotide labelled probe which hybridises to a primate-specific highly repetitive α- satellite D17Z1 locus in the genome – located on chromosome 17

Question 45

whats the best method for DNA quantification?

Answer 45

real-time PCR