MOLBIO - MT 122 - Lecture 4 - Nucleic Acid Extraction Methods

Breaking the cell wall (if present)
Cell lysis

The initial release of the cellular material is achieved by?

Density-gradient centrifugation strategies

Early routine laboratory procedures for DNA isolation were developed from?

a. Semiconservative replication of DNA

Meselson and Stahl used such a method in 1958 to demonstrate?

a. Semiconservative replication of DNA
b. Conservative replication of DNA
c. Precipitated isolation of DNA
d. None of the choices

1869

Miescher first isolated DNA from human cells via precipitation in?

b. First statement is incorrect. Second statement is correct.

Large DNA and proteins CANNOT renature properly when neutralized in acetate at LOW pH after alkaline treatment.

Large (greater than 50 kbp) chromosomal DNA and proteins cannot renature properly when neutralized in acetate at high pH after alkaline treatment, forming large aggregates. The relatively small plasmids return to their supercoiled state and stay in solution.

a. First statement is correct. Second statement is incorrect.
b. First statement is incorrect. Second statement is correct.
c. Both statements are correct
d. Both statements are incorrect.

1- to 50-kb

Alkaline lysis procedures were used extensively for extraction of what range sizes of plasmid DNA during the early days?

TRUE

TRUE OR FALSE

Nucleic acid is routinely isolated from human, fungal, bacterial, and viral sources in the clinical laboratory.

c. Both A and C

The initial steps in nucleic acid isolation depend on the?

a. Nature of the starting material
b. Test method
c. Both A and C
d. None of the choices

c. Both statements are correct

Gram negative bacteria have thinner cell wall that is why they are susceptible of lysis. Mycoplasma pneumoniae which is a cell wall-less bacteria and is easily destroyed or lysed.

a. First statement is correct. Second statement is incorrect.
b. First statement is incorrect. Second statement is correct.
c. Both statements are correct
d. Both statements are incorrect.

TRUE

TRUE OR FALSE

Treatment with detergent (1% sodium dodecyl sulfate) and strong base (0.2 M NaOH) in the presence of Tris base, ethylenediaminetetraacetic acid (EDTA), and glucose can also break bacterial cell walls.

FALSE

By boiling or DNA extracted by alkaline procedures can denature DNA to ssDNA which is not suitable for methods like restriction enzyme analysis that needs dsDNA.

TRUE OR FALSE

DNA extracted by boiling or alkaline (NaOH) procedures are most suitable for methods like restriction enzyme analysis.

FALSE

Used for yeast, filamentous fungi, and gram-POSITIVE bacteria.

TRUE OR FALSE

Commercial reagents designed for isolation of DNA in amplification procedures, such as polymerase chain reaction (PCR), are used for yeast, filamentous fungi, and gram-negative bacteria.

TRUE

TRUE OR FALSE

Viral DNA is held within free viruses or integrated into the host genome along with host DNA.

Blood and bone marrow aspirates

Nucleated cells in suspension uses what specimen?

D. Both A and B

WBCs in blood or bone marrow specimens are purified of red blood cells (RBCs) and other blood components by?

a. Differential density-gradient centrifugation
b. Differential lysis
c. Lysozyme treatment
d. Both A and B
e. Both A and C

Ficoll

Differential density-gradient centrifugation

Whole blood or bone marrow mixed with isotonic saline is overlaid with?

a. First statement is correct. Second statement is incorrect.

HYPOTONIC BUFFER (diluted)

Another method used to isolate nucleated cells takes advantage of the differential osmotic fragility of RBCs and WBCs. Incubation of whole blood or bone marrow in hypertonic buffer.

a. First statement is correct. Second statement is incorrect.
b. First statement is incorrect. Second statement is correct.
c. Both statements are correct
d. Both statements are incorrect.

Exosomes

These are small vesicles (30 to 100 nm in diameter), which form by invagination and budding from the inside of cellular endosome vesicles and are secreted by living cells.

Liquid Biopsy

With the high sensitivity achievable from amplification procedures, these sources of circulating nucleic acids can be detected for purposes of diagnostic and prognostic analyses, known as?

Xylene
(a mixture of three isomers of dimethylbenzene)

Fixed, embedded tissue may be deparaffinized by soaking in?

c. Neutral formalin acetone

Along with 10% buffered, gives good nucleic acid quality and fragment fixative size of 2.0-5.0

Which of these tissue fixatives gives a good nucleic acid quality?

a. Zamboni's
b. Clarke's
c. Neutral formalin acetone
d. Paraformaldehyde
e. None of the choices

FALSE

For organic isolation, this is accomplished using a combination of high salt, LOW pH, and an organic mixture of phenol and chloroform.

TRUE OR FALSE

For organic isolation, this is accomplished using a For organic isolation, this is accomplished using a combination of high salt, high pH, and an organic mixture of phenol and chloroform.

c. Cetyltrimethylammonium bromide

a cationic detergent that efficiently separates DNA from polysaccharide.

The isolation of small amounts of DNA from challenging samples such as fungi can be facilitated by pretreatment with?

a. Isopropanol
b. Formalin-alcohol-acetic acid
c. Cetyltrimethylammonium bromide
d. All of the choices

Biphasic emulsion

What forms when phenol and chloroform are added to the hydrophilic suspension of lysed cells (lysate)?

TRUE

TRUE OR FALSE

At the proper pH, centrifugation will settle the hydrophobic phase on the bottom, with the hydrophilic phase on top.

c. Both statements are correct.

DNA will dissolve in the upper aqueous phase. Amphiphilic components will collect as a white precipitate at the interface between the two layers.

a. First statement is correct. Second statement is incorrect.
b. First statement is incorrect. Second statement is correct.
c. Both statements are correct
d. Both statements are incorrect.

2:1 or 1:1 respectively

The ethyl isopropyl alcohol is added to the upper-phase solution at what ratios in which the DNA forms a solid precipitate?

Glycogen

A biological material derived from mussels.

70% ethanol

Excess salt is removed by rinsing the pelleted nucleic acid in?

Salting out

Inorganic DNA extraction is sometimes called?

TRUE

Same with organic isolation methods: uses High salt conditions and low pH, only difference is it DOES NOT include organic extraction step (e.g. phenol and chloroform).

TRUE OR FALSE

Inorganic isolation makes use of low pH and high-salt conditions to selectively precipitate proteins, leaving the DNA in solution.

Solid phase isolation

A more rapid and comparably effective DNA extraction.

Silica-based products

These products were shown to effectively bind DNA in high-salt conditions.

Chelex

It is a cation-chelating resin that can be used for simple extraction of DNA from minimal samples.

Rapid extraction methods

This method is most commonly used in forensic applications but may also be useful for purification of DNA from clinical samples.

c. Both statements are correct.

Rapid Extraction Methods

A suspension of 10% Chelex resin beads is mixed with the specimen. The cells are lysed by boiling.

a. First statement is correct. Second statement is incorrect.

b. First statement is incorrect. Second statement is correct.

c. Both statements are correct

d. Both statements are incorrect.

Two (First and Second approaches)

1st: Isolate mitochondria DNA via centrifugation

and another second high speed centrifugation is done and lysed with detergent. DNA is precipitated with cold ethanol and resuspended with water.

2nd: Isolate mitochondria DNA via centrifugation and then analyzed within the total DNA background by hybridization or PCR.

In the isolation of mitochondrial DNA, how many approaches can be done from the eukaryotic cell?

-20 degrees Celsius and below

RNA is to be performed at what temperature range?

a. c. b.

RMT

Ribosomal RNA (rRNA : 80-90%)

Messenger RNA (mRNA : 2.5-5%)

Transfer RNA

Give the following order of most to least abundant RNA:
(e.g. a, b, c)

a. Ribosomal RNA
b. Transfer RNA
c. Messenger RNA

Detergent or phenol in the presence of high salt (0.2 to 0.5 M NaCl).

The cell lysis step for RNA isolation is performed in?

Guanidine isothiocyanate (GITC)

This is a strong denaturant of RNases and can be used instead of high-salt buffers.

FALSE

It must be ACIDIC, pH 4 to 6

TRUE OR FALSE

For RNA, the organic phase must be alkalinic.

c. Both statements are correct.

Electrophoresis

Fluorescent dyes such as ethidium bromide or SybrGreen I bind specifically to DNA. They are used to visualize the sample preparation and detect RNA.

a. First statement is correct. Second statement is incorrect.

b. First statement is incorrect. Second statement is correct.

c. Both statements are correct

d. Both statements are incorrect.

Supercoiled plasmid DNA

A good preparation of plasmid DNA will yield a bright signal from?

Two distinct bands

A high-quality preparation of RNA will yield how many bands of ribosomal RNA?

TRUE

TRUE OR FALSE

If these bands are degraded (smeared) or absent, the quality of the RNA in the sample is deemed unacceptable for use in molecular assays.

Densitometry of the band intensities

This gives the most accurate measurement of quantity.

a. First statement is correct. Second statement is incorrect.

It is DIRECTLY proportional.

Spectrophotometry

The relationship of concentration to absorbance is expressed as: A = ϵbc. The absorbance at this wavelength is thus inversely proportional to the concentration of the nucleic acid in the sample.

a. First statement is correct. Second statement is incorrect.

b. First statement is incorrect. Second statement is correct.

c. Both statements are correct

d. Both statements are incorrect.

260 nanometers

In spectrophotometry, nucleic acids absorb light at?

TRUE

TRUE OR FALSE

Since phenol absorbs ultraviolet light at 270 to 275 nm, residual phenol from organic isolation procedures can increase 260 readings, thus should be avoided.

Protein

What is the most likely contaminant in a nucleic acid preparation?

c. Both statements are correct.

Fluorometry

The fluorescence is read in a quartz cuvette. modern procedures use the DNA-specific dye Hoechst 33258.

a. First statement is correct. Second statement is incorrect.

b. First statement is incorrect. Second statement is correct.

c. Both statements are correct

d. Both statements are incorrect.

TRUE

TRUE OR FALSE

RNA does not enhance the fluorescence of the fluorescent dyes and is thus invisible to fluorometric detection.

Fluorometry

For methods that require accurate measurements of low amounts of DNA or RNA (in the range of 10 to 100 ng/mL), which is more preferred, fluorometry or spectrophotometry?

Microfluidics

This system is more automated than standard spectrophotometry or fluorometry with advantages of minimal volume sample (as low as 1 ul) and can test multiple samples simultaneously/