MLS 123 Molecular Biology Lecture 3: Nucleic Acid Extraction Methods Quiz

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Questions and Answers

What is the primary reason for measuring the concentration of nucleic acids at 260 nm?

Nucleic acids absorb UV light at 260 nm, which is close to their maximum absorption. (correct)

Phenol contamination is best detected at 260 nm.

The dilution factor is most easily calculated at 260 nm.

Protein contamination is best detected at 260 nm.

What is the recommended range for the 260-nm/280-nm absorbance ratio for a pure DNA preparation?

2.0 to 2.5

2.0 to 2.3

1.6 to 2.0 (correct)

1.8 to 2.2

What is the recommended step to take if the 260-nm/280-nm ratio is less than 1.6?

Repeat the proteinremoval step of the isolation procedure.

Precipitate the nucleic acid and resuspend it.

Perform column purification of the nucleic acid.

All of the above. (correct)

What is the recommended pH range for accurate determination of nucleic acid purity?

pH 7.5 to 8.5 Signup and view all the answers

What is the sensitivity of the SybrGreen II RNA gel stain?

2 ng/mL Signup and view all the answers

How does the intensity of SybrGreen II fluorescence differ between polyadenylated RNA and total RNA?

The intensity is 20-26% lower with polyadenylated RNA. Signup and view all the answers

What is the main source of nucleic acid in human blood and bone marrow?

White blood cells (WBCs) Signup and view all the answers

Which component of blood is a rich source of proteins, lipids, and other molecules but not nucleic acids?

Serum Signup and view all the answers

What is the main technique used to isolate nucleated cells from blood and bone marrow based on osmotic fragility?

Hypotonic buffer incubation Signup and view all the answers

Which components are released into the bloodstream by solid tumors and transplanted organs?

Exosomes and nucleic acid Signup and view all the answers

What are exosomes and where do they originate from?

Small vesicles from cellular endosomes Signup and view all the answers

Which method is used to concentrate viroids from a solution?

Centrifugation Signup and view all the answers

What is the primary purpose of nucleic acid extraction?

To isolate the target nucleic acid (DNA or RNA) from the cell for use in subsequent procedures. Signup and view all the answers

Which of the following is NOT a crucial step in the nucleic acid extraction process?

Cell lysis Signup and view all the answers

What is the role of differential density-gradient centrifugation or differential lysis in nucleic acid extraction?

To isolate white blood cells (WBCs) from red blood cells (RBCs) in blood or bone marrow specimens. Signup and view all the answers

Which of the following factors can compromise the test results when extracting nucleic acids?

Using an improper collection tube or method Signup and view all the answers

What is the purpose of the cell lysis step in nucleic acid extraction?

To release the nucleic acid from the cell for subsequent procedures. Signup and view all the answers

What is the primary purpose of adding DNase when isolating glycogen?

To break down DNA contamination in the glycogen sample Signup and view all the answers

What is the primary purpose of the organic isolation method using phenol and chloroform?

To increase the purity of the DNA by removing any remaining proteins or lipids Signup and view all the answers

What is the primary purpose of the high-salt and low-pH conditions in the inorganic DNA extraction method?

To selectively precipitate the proteins, leaving the DNA in solution Signup and view all the answers

What is the purpose of the cetyltrimethylammonium bromide (CTAB) pretreatment when isolating DNA from fungi?

To efficiently separate the DNA from polysaccharide contamination Signup and view all the answers

What is the purpose of adding carrier (e.g., 10-20 micrograms per milliliter) to the isopropanol mixture during DNA extraction?

To help precipitate the DNA and improve its recovery Signup and view all the answers

What is the purpose of the centrifugation step in the organic isolation method using phenol and chloroform?

To collect the hydrophobic contaminants, such as lipids and lipoproteins, in the bottom phase Signup and view all the answers

What is the purpose of using Chelex resin in DNA extraction?

To chelate cations Signup and view all the answers

Why must gloves be worn in the RNase-free area during RNA isolation?

To avoid RNase contamination Signup and view all the answers

What is a key feature of the rapid extraction methods mentioned in the text?

Use of methods that provide clean DNA suitable for amplification Signup and view all the answers

Why is it crucial to allocate a separate RNase-free area in the laboratory for RNA analysis?

To avoid RNA degradation due to RNase activity Signup and view all the answers

In the Chelex resin DNA extraction method, what happens after the cells are lysed by boiling?

DNA in the supernatant is cooled and further extracted with chloroform Signup and view all the answers

What distinguishes Chelex resin DNA extraction from traditional phenol-chloroform methods?

Chelex resin does not require phenol or chloroform Signup and view all the answers

Study Notes

Nucleic Acid Contamination and Purity

Phenol contamination must be avoided when measuring concentration at 260 nm, as it absorbs UV light close to the maximum absorption of nucleic acids
Nucleic acid solutions are read at two or three distinct wavelengths to estimate purity
Protein is a common contaminant for DNA determination, and its presence can be detected by the absorbance ratio of 260 nm to 280 nm
A 260-nm/280-nm ratio of less than 1.6 indicates possible protein contamination

RNA Contamination and Interference

RNA can contaminate DNA preparations, leading to interference in DNA detection
RNA can be removed using RNase, and RNA preparations can be treated with RNF DNase to remove DNA contamination
SybrGreen II RNA gel stain can measure RNA, with a sensitivity of 2 ng/mL

Nucleic Acid Extraction Methods

Differential density-gradient centrifugation or differential lysis can be used to purify nucleated cells from blood or bone marrow specimens
Cell lysis is the initial process, followed by purification and concentration determination
The target nucleic acid should be free of contaminants like protein, carbohydrates, lipids, or other nucleic acids

DNA Isolation

DNA can be isolated from humans, fungi, bacteria, and viruses
The isolation method depends on the starting material and the test method
DNase can be used to remove DNA from glycogen preparations
Inorganic isolation methods use low-pH and high-salt conditions to precipitate proteins, leaving DNA in solution
Organic isolation methods use a combination of high salt, low pH, and an organic mixture of phenol and chloroform to dissolve hydrophobic contaminants

RNA Isolation

RNA is labile and requires precautions to avoid sample degradation
RNases must be eliminated or inactivated before isolation to prevent RNA degradation
A separate RNase-free area of the laboratory is necessary for RNA analysis
Gloves must always be worn in the RNase-free area

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Description

Test your knowledge on DNA and RNA extraction methods, isolation processes, and quality measurements of nucleic acids. Explore differential density-gradient centrifugation and other techniques in this quiz.