3. NUCLEIC ACID ISOLATIOON - LEC Flashcards
Release of nucleic acid from the cell
Nucleic acid isolation
The initial release of the cellular material is achieved by
breaking the cell wall and nuclear membranes with cell lysis
The target material is _____ nucleic acid isolation
purified
First isolated DNA from human cells through alkaline lysis method
Miescher 1869
Demonstrate semi-conservative replication of DNA
Meselson and Stahl 1958
Procedures used extensively for extraction of 1-50 kb plasmid DNA from bacteria during the early days of recombinant DNA technology
Alkaline lysis
Expected DNA yield for blood(1ml, 3.5-10 x 10^6 WBs/mL) and buffy coat (1ml blood)
50-200ug
Expected DNA yield for bone marrow (1mL)
100-500ug
Expected DNA yield for cultured cells (10^7 cells)
30-70ug
Expected DNA yield for solid tissue (1mg)
1-10 ug
Expected DNA yield for lavage fluids (10ml)
2-250ug
Expected DNA yield for Mitochondria (10-mg tissue, 10 cells)
1-10ug
Expected DNA yield for plasmid DNA, bacterial culture (100ml overnight culture)
350ug - 1 mg
Expected DNA yield for bacterial culture (0.5mL, 0.7 absorbance units)
10-35ug
Expected DNA yield for feces (1mg; bacteria, fungi)
2-228 ug
Specimens adequate for analysis without DNA amplification
Blood
Buffy coat
Bone marrow
Cultured cells
Solid tissue
Lavage fluids
Mitochondria
Plasmid DNA, bacterial culture
Bacterial culture
Feces
Specimens adequate for analysis with DNA amplification
Serum plasma, CSF
Dried blood
Saliva
Buccal cells
Bone, teeth
Hair follicles
Fixed tissue
Feces
Expected DNA yield for Serum, Plasma, CSF (0.5mL)
0.3-3 ug
Expected DNA yield for dried blood (0.5-1 cm diameter spot)
0.04-0.7 ug
Expected DNA yield for Saliva (1ml)
5-15 ug
Expected DNA yield for buccal cells (1mg)
1-10 ug
Expected DNA yield for bone, teeth (500mg )
30-50 ug
Expected DNA yield for hair follicles
0.1-.0.2 ug
Expected DNA yield for fixed tissue (5-10x10 micron sections ; 10mm)
6-50 u
Expected DNA yield for feces (animal cells, 1mg)
2-100 ug
Blood and bone marrow specimens preferred tube
Yellow tube with ACD
Acid citrate dextrose for molecular studies
Other tubes for blood and bone marrow
Tripotassium / K3 (purple)
Sodium Heparin (brown) and Lithium Heparin (green) - cytogenetics studies
Non-additive tubes (Red) - cell free from specimen
Sample preparation of bacteria and fungi
Enzyme digestion
Alkaline extraction
Mechanical Disruption
Boiling extraction
Proteinase K: digests proteins
Lysozyme digests other cell organelles
gentle procedure
Enzymatic digestion
Most common way of preparing bacteria and fungi samples
1% sodium dodecyl sulfate and 0.2 M NaOH
EDTA as chelating reagent
Glucose for destroying the cell wall
Alkaline Extraction
not usually used as it may also destroy the nucleic acid
grinding if it is solid
glass beads with vigorous shaking if plasmid
Mechanical Disruption
Method used if the sample is treated with lysoenzyme
Diluted sucrose
Triton X-100 detergent
Tris buffer
Edta
Boiling extraction
Sample preparation for nucleated cells in suspension
Differential density-gradient centrifugation
Differential osmotic fragility of RBCs and WBCs
Whole blood or bone marrow mixed with isotonic saline is overlaid with Ficoll
preferred method since it does not penetrate the cell membrane
Differential density-gradient centrifugation
Incubation in hypotonic water will result in the lysis of RBC and WBC
Differential osmotic fragility of RBCs and WBCs
released from solid tumors and transplanted organs
Exosomes
Sources of circulating nucleic acids
Isolation of cell-free nucleic acid requires procedures to concentrate the target nucleic acid before isolation
use of plasma for the purpose of diagnostic and prognostic analysis
Liquid biopsy
Liquid biopsy sources
Plasma, CSF, ascites, pleural fluid
Extracellular viruses is detected with
Liquid Biopsy
Sample preparation for tissue samples
Frozen tissue
Grinding
Mincing
Fixed tissue
least damaging among tested fixatives
Neutral buffer formalin
worst fixatives for DNA recovery
Mercury based fixatives such as Bouin’s and B5
How many base pairs can be obtained from a fixed tissue
100 base pairs
Frozen tissue can be grinded in
liquid nitrogen and homogenizing tissue
what do we use for deparaffinization
xylene and xylol
DNA isolation method
- DNA Isolation chemistries
- Proteolytic Lysis of Fixed Material
- Rapid Extraction Methods
- Isolation of Mitochondrial DNA
- DNA Isolation chemistries
a. Organic Isolation Methods
b. Inorganic Isolation Methods
c. Solid-Phase Isolation
Not used organic isolation method today since they are carcinogenic
Phenol and chloroform
Isolation of small amounts of DNA from challenging samples such as fungi can be facilitated by pre-treatment with
Cetyltrimethylammonium bromide CTAB
Detergent that will separate the polysaccharide from the DNA (chitin)
Cetyltrimethylammonium bromide CTAB
can be added in first step or last step of the procedure (organic isolation method)
- Purpose in the 1st step: to remove the presence of RNA
- Purpose in the last step: to remove residual RNA
RNAse
dissolves hydrophobic
contaminants such as lipids and lypoproteins
Phenol and Chloroform
Most preferred salt for organic isolation method
Sodium acetate and sodium chloride
Alternative salt for organic isolation method
Potassium acetate and lithium chloride
Procedure of organic isolation method
a. Lysis
b. Acidification
c. Centrifuge
d. Extraction
e. Precipitation
f. The DNA at the bottom still has residual salts
g. Resuspend them in buffer, Tris-EDTA or distilled water .
Organic isolation uses ________ for lysis
Sodium hydroxide and SDS
What do we use for acidification
Acetic acid and Salt
In what step will we add equal amount of phenol and chloroform
Extraction
In extraction we will form 3 layers which are the
> Aqueous phase: hydrophilic components
> Ampiphilic phase: both hydrophobic & hydrophilic components
> Organic phase: lipids & hydrophobic organic elements.
What will we use to precipitate the DNA
ethanol
This method was developed due to dangers of organic isolation
Inorganic isolation
Inorganic isolation main disadvantage
Salt precipitates protein and not other contaminants
high salt solution is used (Na acetate, NaCl, potassium acetate, lithium chloride)
Also called as “SALTING OUT”
Preparation of Inorganic isolation Method
method that uses silica based products
more rapid
Solid phase isolation
source of silica
Diatomaceous earth
Commonly used to isolate viral and bacterial DNA from serum,
plasma, or cerebrospinal fluid.
Solid phase isolation
Preparation of solid phase isolation
a. 1st washing adsorption
b. 2nd washing - elution
Proteolytic Lysis of fixed materials procedure
a. use xylene to remove the wax
b. use 70% ethanol to rehydrate
c. place the sample in a Tris-EDTA buffer
d. add proteinase K to lyse the proteins in the sample
Cation-chelating resin
chelex
A suspension of 10% resin beads is mixed with the specimen,
and the cells are lysed by boiling
Rapid extraction method
This method is most commonly used in forensics and
purification of DNA
Rapid Extraction Method
Isolation of Mitochondrial DNA two types
a. Centrifugation (2-step centrifugation)
b. Isolation of total DNA
speed of the first step of centrifugation in isolating mitochondrial DNA
700 to 2600 x g
What is the purpose of the first centrifugation step in mitochondrial DNA isolation?
Separate cell debris from supernatant containing mitochondria.
What is produced after the first low-speed centrifugation?
lower layer (cell debris), upper layer (supernatant).
What does the second high-speed centrifugation step yield?
Mitochondrial pellet and supernatant
Why is 1% SDS detergent used in mitochondrial DNA isolation?
To lyse mitochondria and remove proteins.
What is the role of cold ethanol in mitochondrial DNA isolation?
To precipitate mitochondrial DNA.
What buffer is used to resuspend mitochondrial DNA after precipitation?
Tris-EDTA buffer.
What method is used for total DNA isolation during mitochondrial DNA extraction?
Chemical extraction or proteolytic lysis
Why must RNases be eliminated during RNA isolation?
RNases degrade RNA, so they must be eliminated or inactivated.
What temperatures are ineffective in deactivating RNases?
-20°C, 100°C, and 121°C do not deactivate RNases.
What temperature is required to decontaminate glassware for RNA isolation?
Glassware must be decontaminated at 400°C for four to six hours.
What are the types of RNA found in total RNA?
rRNA, mRNA, and tRNA.
What are RNase-free (RNF) conditions?
Conditions where RNase enzymes are eliminated to prevent RNA degradation.
What is the RNA yield from 1 mL of blood?
1-10 µg.
What is the RNA yield from 1 mL of buffy coat ?
5-10 µg.
What is the RNA yield from 1 mL of bone marrow?
50-200 µg.
What is the RNA yield from 10^7 cultured cells?
50-150 µg.
How should RNA samples be stored to prevent degradation?
Frozen in liquid nitrogen or immersed in a buffer that inactivates RNases.
What is the RNA yield from 1 mg of buccal cells?
1-10 µg.
What is the RNA yield from 1mm2 of fixed tissue?
0.2-3 µg.
What is the RNA yield from 1 mg of solid tissue?
0.5-4 ug
What is the RNA yield from 0.5ml of bacterial culture?
10-100 ug
Which sample type is preferred for human RNA testing?
Bone marrow.
Can viral RNA be isolated from cell-free fluids?
Yes, using spin columns or beads.
RNA isolation methods
Organic Isolation
Solid Phase Isolation
Proteolytic lysis and Fixed material
Isolation of PpolyA (messenger) RNA
What is used for RNA organic isolation?
Detergent or phenol with high salt (0.2 to 0.5 M NaCl).
What are the RNase inhibitors used in RNA isolation?
Guanidine isothiocyanate (GITC) and 2-mercaptoethanol.
What is the ratio of reagents in organic RNA extraction?
25:24:1 (acid phenol:chloroform alcohol).
What is the role of chloroform in RNA isolation?
Enhances nucleic acid extraction by protein denaturation and phase separation.
What is the pH of the organic phase in RNA isolation?
pH 4-6 (acidic).
What is used to optimize RNA adsorption in solid-phase isolation?
Commercial reagents supplied with silica-based columns
What type of RNA does the solid matrix in RNA isolation adsorb?
Single-stranded RNA.
Fixatives used in extraction of RNA
10% buffered neutral formullin
Acetone
Zambionis
Clarke’s
Paraformaldehyde
Methacam
Formalin-alcohol
acetic acid
Carmoy’s
Zenkar’s
Boun’s
What is the desirability and size range of 10% Buffered Neutral Formalin?
Desirability: Good
Size Range: 20-50 kh
What is the desirability and size range of Acetone?
Desirability: Good
Size Range: 20-50 kh
What is the desirability and size range of Zambionis?
Desirability: Not as good
Size Range: 02-20 kh
What is the desirability and size range of Clarke’s?
Desirability: Not as good
Size Range: 08-10 kh
What is the desirability and size range of Paraformaldehyde?
Desirability: Not as good
Size Range: 02-50 kh
Question: What is the desirability and size range of Methacam?
Desirability: Not as good
Size Range: 07-15 kh
What is the desirability and size range of Formalin-Alcohol?
Desirability: Not as good
Size Range: 10-40 kh
What is the desirability and size range of Acetic Acid?
Desirability: Less desirable
Size Range: 01 kh
What is the desirability and size range of Carmoy’s?
Desirability: Less desirable
Size Range: 07-15 kh
What is the desirability and size range of Zenkar’s?
Desirability: Less desirable
Size Range: 07-15 kh
What is the desirability and size range of Boun’s?
Desirability: Less durable
Size Range: 01 kh
Why is polyA RNA important?
It is important in gene expression.
What information does polyA RNA carry?
It carries information from the DNA.
Why is polyA RNA used instead of rRNA in isolation?
It is used instead of rRNA.
How does the isolation process affect the yield of mRNA?
Enrich the yield of mRNA.
What type of oligomers are used in the isolation of polyA RNA?
Uses single-stranded oligomers of thymine or uracil.
How does the polyA tail interact with oligomers in the isolation process?
PolyA tail binds with polyT or polyU oligomers.
How is polyA RNA eluted from the column?
PolyA RNA is eluted by washing the column with warmed, low-salt buffer.
How much polyA RNA is expected from 1 µg of total RNA?
1 µg of total RNA = 30-40 ng of polyA RNA.
How does the isolation process reduce degradation of RNA?
Lessens degradation by degrading nucleases
Measurement of Nucleic Acid- Quality and
Quantity
- Electrophoresis
- Spectrophotometry
- Fluorometry
- Microfluidics
What is the purpose of using dyes in electrophoresis?
Uses dyes to visualize the sample preparation.
What types of nucleic acids does Ethidium Bromide stain?
both DNA and RNA
What does SybrGreen I stain?
Stains DNA.
What does SybrGreen II stain?
Stains RNA.
What is unique about Silver Stain compared to fluorescence stains, and how does its storage affect staining?
Not a fluorescence stain; longer storage turns all staining “red”.
Which dyes are used for DNA and RNA, and which is used for visual inspection?
Ethidium Bromide and SybrGreen I are used for DNA and RNA, while Silver Stain is used for visual inspection.
What does a bright signal indicate in the context of supercoiled plasmid DNA?
Bright signal from supercoiled plasmid DNA.
Where does high-molecular-weight chromosomal DNA typically appear on the gel?
Near top of the gel.
What are the two distinct bands of ribosomal RNA observed in RNA electrophoresis?
28S rRNA
18S rRNA
How is the concentration of a sample determined using fluorescent dyes in electrophoresis?
Fluorescent dyes quantified by the fluorescence intensity of the sample aliquot run on the gel; higher intensity = higher concentration.
Why is densitometry considered more accurate for measuring band intensity in electrophoresis?
More accurate since a standard measure of density is provided.
At what wavelength do nucleic acids absorb light?
Nucleic acids absorb light at 260 nm.
What principle does spectrophotometry follow?
Spectrophotometry follows the Beer-Lambert Law.
What are the absorptivity constants for double-stranded DNA and RNA?
50 = dsDNA
40 = RNA
What is the concentration equivalent of one optical density unit at 260 nm for double-stranded DNA and RNA?
One optical density unit at 260 nm is equivalent to:
50 mg/L (or 50 µg/mL) of double-stranded DNA
40 µg/mL of RNA
How do you calculate the DNA concentration from an absorbance reading of 0.200 and a 1/100 dilution?
A DNA preparation diluted 1/100 yields an absorbance reading of 0.200 at 260 nm. Multiply:
0.200 absorbance units × 50 µg/mL per absorbance unit × 100 = 1,000 µg/mL
How do you calculate the yield of DNA if the concentration is 1,000 µg/mL and the volume is 0.5 mL?
The yield is calculated using the DNA concentration and volume.
1,000 µg/mL × 0.5 mL = 500 µg
At what wavelength do organic compounds show peak absorbance?
Peak absorbance at 230 nm.
At what wavelength does phenol show peak absorbance?
Peak absorbance at 270 nm.
At what wavelength do proteins show peak absorbance?
Peak absorbance at 280 nm.
At what wavelength does particulate matter show peak absorbance?
Peak absorbance at >330 nm.
What does 3,5-diaminobenzoic acid 2HCI (DABA) bind to?
Binds to all deoxyribose such as dsDNA & ssDNA.
Which base pairs does Hoechst 33258 bind to?
Binds to adenine & thymine base pairs.
What is the detection limit of DNA using Hoechst 33258
Can detect 250 µg/mL of DNA.
Used for specifically detecting dna
Hoechst 33258
What technology is utilized in microfluidics?
Uses lab-on-a-chip technology.
Where is the sample applied in microfluidics?
Sample is applied to a multi-well chip.
How does the sample move in a microfluidic system?
Sample moves through microchannels across a detector.
What configurations can the instrument software generate in microfluidics?
Instrument software generates images in electropherogram (peak) or gel (band) configurations.
