nucleic acid isolation (part 2 of exam 1)

density-gradient centrifugation

relies on solubility differences among cellular components

step by step DNA extraction

breakdown cell wall/membrane

digest proteins

remove supernatant

precipitate DNA and spin down

reconstitute isolated DNA

regardless of specimen, what must be done to the cell?

lysis

why is cell lysis critical

liberates nucleic acids

lysis buffer

lyses nuclear and cellular membranes

maintains pH

maintains DNA integrity

separates DNA from cell debris

lysis buffer components

1% SDS (detergent)

tris (buffer)

EDTA (chelating agent)

what are some other ingredients sometimes added to lysis buffer?

0.2 M NaOH

proteinase K

how are microorganisms lysed?

high pH and detergents

what kind of cell walls do microorganisms have?

tougher cell wells, require enzymes that digest polymers

mechanical lysis

grinding w/ glass beads or sonication

where do the nucleic acids come from in blood or bone marrow?

white blood cells

how do you purify the white blood cells when you have blood/bone marrow?

remove red blood cells via differential density-gradient centrifugation or differential lysis

differential density-gradient centrifugation

cell suspension in isotonic saline suspension with Ficoll gradient

before centrifugation, Ficoll is between red and white cells

after centrifugation, white cells are above Ficoll

differential lysis

use of differential osmotic fragility to get rid of red blood cells, then centrifugation

what is Ficoll?

highly branched sucrose polymer

capillary blood collection

collected from heel sticks and often used to assess for congenital diseases

extraction of nucleic acids from tissue samples

tissue samples dissociated before DNA isolation

fixation, embedding, sometimes freezing, buffers

what is the least damaging fixative agent?

buffered formalin

DNA isolation methods

organic, inorganic, solid-phase

organic isolation of DNA

high salt, low pH, and phenol + chloroform

use RNAase

organic isolation of DNA workflow

cell lysis

acidification

extraction

precipitation

rehydration and storage

inorganic isolation of DNA (“salting out”)

avoids use of phenols

use high salt, low pH to precipitate proteins and leave DNA

solid-phase isolation of DNA (most used)

uses solid matrix via column/binds that bind and hold DNA for washing

how is mitochondrial DNA (mtDNA) inherited?

maternally

how much does total DNA in a cell does mitochondrial DNA make up?

1%

why would we want to isolate mtDNA?

phylogenetics

evolutionary biology

anthropology

pathogenesis

two approaches to isolation of mtDNA

• isolate mitochondria by centrifugation

• isolate mitochondria from total DNA

isolating mitchondria by centrifugation is similar to what type of method?

organic isolation

how can you isolate mitochondria from total DNA?

PCR to amplify mtDNA

why would you want to isolate RNA?

gene expression analyses (e.g in genetic imprinting)

phylogenetic and population analyses

why should you work on ice when isolating RNA?

RNA is unstable in its single-stranded form

precautions needed to prevent RNA degradation

RNases

RNase inhibitors to clean work area

have a separate RNase-free area in lab

diethyl pyrocarbonate (DEPC) water

inactivates enzymes that are present in RNA

how much of total RNA is rRNA?

80-90%

how much of total RNA is mRNA?

2.5-5%

organic isolation of RNA

cell lysis (w/ high salt and RNase inhibitors)

proteins extracted organically w/ acid phenol:chloroform:isoamyl alcohol

what can be used to denature RNases?

guanidine isothiocyanate (GITC)

what part of the tube contains the RNA in organic isolation?

upper aqueous phase

what does chloroform do in organic isolation of RNA?

denatures proteins

solid-phase isolation of RNA

use filter columns

isolation of polyA RNA (mRNA)

wash away residual RNA and polyA RNA is eluted w/ low-salt buffer containing detergent

30-40 ng of mRNA can be obtained from 1 ug of total RNA

after extraction, what should you look at?

is it there?

quality and quantity of nucleic acid extracted

what can you use to analyze quality of nucleic acid extracted?

electrophoresis (visualize bands)

dyes for visualization of nucleic acids

ethidium bromide (EtBR)

SYBR Green
SYBR Gold

Silver stain

ethidium bromide

intercalating agent - inserts itself btwn bp in double helix

glows orange under UV light

detects both DNA and RNA

SYBR Green

binds the grooves rather than intercalating

greater sensitivity than EtBr

at what wavelength do nucleic acids absorb light?

260 nm through adenine residues

Beer-Lambert law definition

used to calculate concentration

Beer-Lambert law equation

A = ebc

A = absorbance

e = molar absorptivity (L/mol cm)

b = path length (cm)

c = concentration (mg/L)

absorptivity constants

50 ug/mL for dsDNA

40 ug/mL for RNA

how to calculate concentration of DNA?

DNA concentration = 50 X Absorbance

units are ug/mL

estimating nucleic acid purity (you ate proteins)

A260/A280 ratio

used to assess purity of DNA and RNA

what ratio is accepted as pure DNA?

1.8

(lower ratios = less than ideal purity)

what ratio is accepted as pure RNA?

2.0

secondary measure of nucleic acid purity

A260/A230

fluorometry

measures fluorescence related to DNA or RNA in association w/ nucleic acid-specific fluorescent dyes

often used where accurate measurements of DNA is critical