MOLBIO LEC - Nucleic Acid Extraction Methods

Release the nucleic acid from the cell

Purpose of nucleic acid extraction:

True

T or F: The target nucleic acid should be free of protein, carbohydrates, lipids, or other nucleic acids

Breaking the cell wall; Cell lysis

How is the initial release of cellular material achieved?

Purified

Following gentle lysis of a cell, the target material must be ____ before concentration and purity are determined

Density-Gradient Centrifugation Strategies

Early routine laboratory procedures for DNA isolation was developed from what?

Large aggregates (precipitation)

Large chromosomal DNA and proteins cannot renature properly, and instead they form ____

Supercoiled state

Relatively small plasmids return to their ____

1-50 kb

Alkaline lysis procedures were used only for ____ plasmid DNA from bacteria

True

T or F: Test validation should include sample-collection methods due to the diversity of sample types and nucleic acid sources

Gram-negative bacteria

Early recombinant DNA experiments we performed with this type of bacteria due to its thin wall; susceptible to lysis

Glass beads

Cell walls may also be broken mechanically by grinding or mixing with ____

Gentler Enzymatic Methods

These are preferred methods for involving larger chromosomal targets

1% Sodium dodecyl sulfate

What detergent may be used in isolating bacteria/fungi?

0.2M NaOH

What strong base may be used in isolating bacteria/fungi?

Boiling or Alkaline procedures

DNA extracted by ____ or ____ results in denatured DNA, and single stranded DNA may not be suitable for restriction enzyme analysis

Commercial reagents

What can be used to lyse cell walls for yeast, filamentous fungi, or gram-positive bacteria?

Viral DNA

Held within free viruses or integrated into the host genome

Plasma

What specimen is used for viral detection?

Blood and Bone Marrow Aspirates

What specimen/s can be used for nucleated cells in suspension?

White Blood Cells

Nucleic acids in blood and bone marrow come from?

Anticoagulants

Use of these may trap WBCs when preventing clotting

Differential Density-Gradient Centrifugation

How are WBCs purified of RBCs and other blood components?

Ficoll

In DDGC, whole blood or bone marrow in an isotonic solution is overlaid with ____

Liquid Biopsy

When using a plasma specimen, proteins, lipids, and other nucleic acid may interfere from tumors, organs, or shed cells. What high sensitivity test can detect the circulating nucleic acids for diagnosis and prognosis?

Concentrate

In liquid biopsies, isolation of cell-free nucleic acid requires procedures to ____ the target nucleic acid before isolation

Solid-Phase Collection (beads/columns)

How are cell-free nucleic acids concentrated?

Dissociated

Tissue samples must be ____ before DNA isolation

Xylene

Fixed, embedded tissue may be deparaffinized by soaking it in what?

Buffered Formalin

This is the least damaging tissue fixative, and tissue specimen may be used without dewaxing

Bouin's and B-5

Fixative/s worst for DNA recovery

Ethanol

After xylene, tissue is rehydrated in decreasing concentrations of ____

100 base pairs

DNA target products of ____ or less can be obtained from fixed tissue

Proteinase K

Extended digestion of ____ may yield longer fragments

Organic Isolation Methods of DNA

- Purification of lipids and lipoproteins with high salt, low-ph, phenol, & chloroform

- Pretreatment with cetyltrimethylammonium bromide to separate DNA

- DNA in upper phase is precipitated by mixing ethanol/isopropanol salt (2:1 or 1:1)

- Carrier molecules used to recover minimal DNA

- Glycogen for coprecipitation

Inorganic Isolation Methods of DNA

- aka Salting Out

- low-pH and high salt to precipitate proteins

- isopropanol precipitates DNA

- resuspended in TE buffer/water

Solid-Phase Isolation of DNA

- more rapid

- solid matrices bind DNA (columns or beads)

- special buffers are used to lyse bacteria, fungi,etc.

- cell lysate is added to column

- carrier RNA or DNA enhance recovery (>200 nucleotides)

- elute with water/TE

- magnetic beads used to bind DNA

Spin Columns

These are placed in microcentrifuge tubes to isolate viral and bacterial DNA from sample; routinely used for DNA isolation in genetics and oncology

Proteolytic Lysis of Fixed Material of DNA

- large screening numbers of samples

- limited amounts of starting material

- challenging samples

- cells lysed in detergents (SDS or Triton)

- proteinase K lyses cells

- released DNA can be used directly in amplification

True

T or F: Fixed tissue is a more conveniently accessed patient material, but it is a challenging sample

Rapid Extraction Methods

- most used method in forensics

- minimal sample requirement; allows use of material previously not utilizable

- DNA extraction/storage cards provide sufficiently clean DNA for amplification

- 10% chelex resin beads lyses cells by boiling

- DNA extracted with chloroform

Isolate Mitochondria (centrifugation)
Isolate total DNA

Isolation of Mitochondrial DNA methods:

Isolate Mitochondria by Centrifugation

- cell prep. homogenized by grinding on ice

- homogenate centrifuged at 700-2600 x g

- mitochondria pelleted at 10,000–16,000 x g

- lyse with detergent

- lysate and proteinase remove contaminants

- mitochondrial DNA precipitated with cold ethanol

Isolate Total DNA

The preparation will contain mitochondrial DNA that can be analyzed within the total DNA background by hybridization or PCR

RNases

- These enzymes are small proteins that can renature, even after autoclaving, and regain activity
- They remain active @ temperatured down to 20C and below

False

T or F: Like DNases, RNases can stay activated before isolation of RNA

ribosomal RNA

Most abundant RNA in all cells at 80-90%

Agarose Gel Electrophoresis

rRNA consists of large and small components, which are visualized by?

messenger RNA

The next most abundant RNA fraction at 2.5-5%

True

T or F: mRNA can be faintly detected in agarose gel electrophoresis

Transfer RNA and small nuclear RNAs

Apart from rRNA and mRNA, what else makes up the total RNA sample?

Array analysis or Quantitative Reverse Transcriptase PCR

Examples of RNA tests that involve gene expression

Reticulocytes

In RNA isolation chemistries, ____ in blood and bone marrow are lysed by osmosis or separated from WBCs by centrifugation

True

T or F: In dissociation, keep sample frozen in liquid nitrogen or immersed in buffer that will inactivate intracellular RNases

Organic Isolation of RNA

- cell lysis by detergent or phenol with high salt (NaCl) or RNase inhibitors

- phenol:chloroform:isoamyl alcohol (25:24:1) to extract RNA

- upper phase with RNA is extracted and precipitated with ethanol (2) or isopropanol (1)

- RNA precipiate washed in 70% ethanol and resuspended in RNF buffer

Guanidine Isothiocyanate

Strong denaturant of RNases can be used instead of high-salt buffers

2-mercaptoethanol

A strong reducing agent can be added in organic isolation of RNA during cell lysis

Chloroform

This enhances the extraction of nucleic acids by denaturing proteins and promoting phase separation

Isoamyl Alcohol

This is added to prevent foaming

Acidic

For RNA, organic phase must be ACIDIC/BASIC?

Solid-Phase Isolation of RNA

- strong denaturing buffer conditions must be adjusted before addition of lysate

- ethanol may be added

- filter column removes particulate material

- commercial reagents optimize RNA adsorption

- lysate added in high-salt chaotropic buffer

- eluant suspended by gravity

Isolation of polyA mRNA

- single-stranded oligomers of thymine or uracil immobilized on a matrix resin column/beads are used to enrich mRNA yield
- polyT or polyU will bind to poly A on mRNA
- polyA RNA is eluted

30-40 ng mRNA

How much mRNA can be obtained from 1 ug of total RNA?

Genes

Majority of mRNA consist of mRNA highly expressed in ____

Ethidium Bromide or SybrGreen I

These fluorescent dyes bind to DNA and are used to visualize sample preparation

True

T or F: A good preparation of plasmid DNA will yield a bright signal from supercoiled plasmid DNA with minor or no other bands that represent nicked or broken plasmid

High MW Chromosomal DNA

Should collect as a bright band near the top of the gel in electrophoresis

2

High-quality Preparation of RNA will yield how many bands of rRNA?

Densitometry

The ____ of the band intensities gives the most accurate measurement of quantity

260 nm

Nucleic acid absorbs light at ____ through the adenine residues

Beer-Lambert Law

Using this, concentration can be determined from the absorptivity constants

50; 40

Absorptivity constants of DNA ____ and RNA ____

Directly Proportional

Absorbance at 260 nm is DIRECTLY/INVERSELY proportional to the concentration of nucleic acid in the sample

Phenol

This absorbs UV light at 270 to 275 nm, close to the wavelength maximum absorption by nucleic acids

True

T or F: Residual phenol from organic isolation procedures can increase 260 readings; avoid phenol contamination

Protein

Due to its abundance and close association with nucleic acid, what is the most likely contaminant in a nucleic acid preparation?

280 nm

Protein absorbs light at ____ through tryptophan and tyrosine residues

3,5-diaminobenzoic acid 2HCl

Early methods of fluorometry uses this fluorescent dye

Intact DNA

Fluorometry may be less convenient but is recommended for procedures like accurate measurement of ____

Hoechst 33258

More modern fluorometry procedures use this DNA-specific dye

2 hours

Once dye and sample solution are mixed, fluorescence must be read within ____ because this is the time the sample is stable

Quartz Cuvette

What type of cuvette is fluorescence read in?

True

T or F: Most laboratories use spectrophotometry because samples can be read directly, but fluorometry may be preferred for accurate measurements of low amounts of DNA

Microfluidics

This encompasses "lab-on-a-chip technology" for nucleic acid quantification

RNA Integrity Number

In microfluidics, a quantification estimate for RNA called ____ is determined as a standard measure of RNA integrity

1 uL

Microfluidics is more automated than standard spectrophotometry or fluometry and only requires a minimal volume of ____ to test multiple samples simultaneously

MicroRNAs and Gene expression

Microfluidics is useful for the analysis of ____ in eukaryotes and ____ in bacteria