Nucleic Acid Purification and Analysis Quiz

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Questions and Answers

What is the extinction coefficient used to calculate the concentration of single-stranded DNA (ssDNA) at 260 nm?

33 µg/mL (correct)

50 µg/mL

40 µg/mL

60 µg/mL

Which form of RNA makes up the majority of total RNA samples in an organism?

Small nuclear RNA (snRNA)

Messenger RNA (mRNA)

Ribosomal RNA (rRNA) (correct)

Transfer RNA (tRNA)

What is the primary method for estimating DNA and RNA concentrations that involves measuring light absorption?

Spectrophotometry (correct)

Fluorometry

Chromatography

Electrophoresis

In the Lambert-Beer Law, if the absorbance at 260 nm (A260) is known, what can be determined?

Molar concentration [M] Signup and view all the answers

What is the expected outcome in gel electrophoresis for total RNA preps?

Two prominent bands representing rRNA Signup and view all the answers

What is the primary purpose of adding organic solvents during nucleic acid purification?

To separate water-soluble molecules from lipids and proteins Signup and view all the answers

Which component remains in the aqueous phase under acidic conditions during nucleic acid extraction?

RNA Signup and view all the answers

What technique is primarily used after lysis with chaotropic salt for nucleic acid purification?

Spin column chromatography with a silica matrix Signup and view all the answers

What is the role of DNase and RNase in the nucleic acid purification process?

To remove unwanted nucleic acids from the sample Signup and view all the answers

What additional step may be necessary after nuclease treatment in nucleic acid purification?

Phenol/chloroform extraction for protein removal Signup and view all the answers

Who first established the standard method for nucleic acid purification that involves phenol/chloroform extraction?

Chomczynski and Sacchi Signup and view all the answers

What type of molecules are primarily found in the organic phase after nucleic acid extraction?

Lipids and proteins Signup and view all the answers

Which of the following statements accurately describes the solution after centrifugation in nucleic acid extraction?

Aqueous phase contains nucleic acids and water-soluble molecules Signup and view all the answers

What is the role of reverse transcriptase in generating cDNA?

To convert mRNA into complementary DNA Signup and view all the answers

In quantitative PCR, what feature does SyBr Green bind to?

Minor groove of double-stranded DNA Signup and view all the answers

Which step follows the generation of clusters in second generation sequencing?

Sequencing by synthesis Signup and view all the answers

What is the primary purpose of a melting curve analysis in quantitative PCR?

To assess the specificity of the amplified product Signup and view all the answers

Which of the following is a characteristic of second generation sequencing techniques?

It utilizes sequencing by synthesis for data acquisition Signup and view all the answers

How does cDNA differ from genomic DNA?

cDNA is synthesized from RNA, whereas genomic DNA is not Signup and view all the answers

Which of the following components is essential for sequencing by synthesis in second generation sequencing?

DNA polymerase Signup and view all the answers

What aspect of the cDNA synthesis process involves a poly(A) tail?

It aids in the selection of mRNA during cDNA synthesis Signup and view all the answers

What is the maximum fragment size that can typically be sequenced in highly complex libraries?

150 bp Signup and view all the answers

Which of the following is a benefit of using Nanopore sequencing?

Direct identification of modified bases Signup and view all the answers

What is a significant disadvantage of Nanopore sequencing?

Pore clogging and quality of the nucleic acid library Signup and view all the answers

Which instruments are associated with third generation sequencing technologies?

GridION Signup and view all the answers

Which of the following statements about short read sequencing is accurate?

It can fully sequence fragments of up to 150 bp for highly complex libraries. Signup and view all the answers

What is the first step involved in the preparation of sequencing libraries?

Fragment shearing Signup and view all the answers

What characteristic does Nanopore sequencing improve significantly?

Haplotype assembly Signup and view all the answers

How does the motor protein helicase contribute to Nanopore sequencing?

It unwinds DNA strands for long-read sequencing. Signup and view all the answers

What primary purpose does a Southern blot serve in molecular biology?

To analyze gene targeting success Signup and view all the answers

Which method is utilized for RNA detection in tissue samples?

Northern blot Signup and view all the answers

What is the role of Taq polymerase in the PCR process?

It extends the primer to form new DNA strands Signup and view all the answers

Which technique relies on fluorescent dyes for nucleic acid detection?

Fluorescent in situ hybridization Signup and view all the answers

What is a key characteristic of the PCR process?

It generates identical copies of a specific DNA segment Signup and view all the answers

What does fluorescent in situ hybridization (FISH) diagnose?

Chromosome translocations Signup and view all the answers

Which of the following is not a step involved in a basic PCR cycle?

Gel electrophoresis Signup and view all the answers

In what context would a Northern blot be used?

To identify RNA size and quantity Signup and view all the answers

Which component is essential for the denaturation step in PCR?

Heat Signup and view all the answers

Which nucleic acid visualization technique is commonly used for chromosome painting?

Fluorescent in situ hybridization Signup and view all the answers

Study Notes

Nucleic Acids

Nucleic acids are composed of nucleotides, which consist of a sugar, a phosphate group, and a nitrogenous base.
RNA (ribonucleic acid) contains ribose sugar, and DNA (deoxyribonucleic acid) contains deoxyribose sugar.
The nitrogenous bases in DNA are adenine, guanine, cytosine, and thymine.
The nitrogenous bases in RNA are adenine, guanine, cytosine, and uracil.

Nucleic Acid Purification: Extraction with Organic Solvents

Nucleic acids are purified from crude lysates using organic solvents (e.g., phenol, chloroform).
Water-soluble molecules (nucleic acids) remain in the aqueous phase, while proteins and lipids are extracted into the organic phase.
Debris is trapped in the interphase between these layers.
RNA remains in the aqueous phase under acidic conditions, while DNA partitions into the interphase.

Nucleic Acid Purification: Spin Column Chromatography with Silica Matrix

Nucleic acids are purified using spin columns with a silica matrix.
DNA and RNA are bound to the silica membrane under high-salt conditions.
Contaminants are washed away with high-salt or ethanolic conditions.
DNA and RNA are eluted in a low-salt buffer or water.

Removing Unwanted Nucleic Acids

Unwanted nucleic acids (DNA or RNA) can be removed by adding nucleases (DNase or RNase) to the sample.
Depending on application, protein removal following nuclease treatment may be necessary (e.g., with phenol/chloroform extraction).

Nucleic Acid Quantification: Spectrophotometry - NanoDrop

Nucleic acid concentration is estimated by measuring light absorption at specific wavelengths (e.g.~260 nm for DNA and RNA).
Different types of nucleic acids (dsDNA, ssDNA, ssRNA) have different extinction coefficients at 260 nm.
The Lambert-Beer Law (A = εbc) is used to calculate the concentration.

Nucleic Acid Quantification using Fluorescent Dyes: Fluorometry (e.g., Qubit)

Fluorescent dyes specific to nucleic acids are used for quantification.
The concentration is correlated to fluorescence intensity.
This method eliminates issues with turbidity.

Nucleic Acid Quality Control: RNA

Ribosomal RNA (rRNA) is a major component of total RNA.
Total RNA preparations should exhibit prominent ribosomal RNA bands on gel electrophoresis.
RNA quality is assessed through different assays. (e.g. number of genes for each polymerase, transcriptional activity, and rRNA abundances).

Nucleic Acid Quality Control: Chip Electrophoresis (e.g., BioAnalyzer, TapeStation)

Chip electrophoresis separates nucleic acid fragments, allowing analysis of RNA or DNA quality.

Nucleic Acid Visualization/Detection:

Fluorescent dyes (e.g., EtBr, SYBR family) are used to visualize nucleic acids on agarose gels.

Nucleic Acid Visualization/Detection: Northern/Southern Blot

Northern blots detect RNA using a labeled DNA probe.
Southern blots detect DNA using a labeled DNA probe.
These methods are used to see the size and abundance of target DNA/RNA molecules in different tissues.

Nucleic Acid Visualization/Detection: Fluorescent in situ hybridization (FISH)

Uses fluorescent probes that bind to specific DNA sequences.
This technique is used to visualize chromosomes and detect chromosomal abnormalities.

PCR (Polymerase Chain Reaction)

PCR amplifies specific DNA sequences through multiple cycles of denaturation, annealing, and extension.
Key component is a thermostable DNA polymerase like Taq polymerase.

Generating cDNA

cDNA is synthesized from mRNA via reverse transcriptase.
This step is necessary for studying gene expression.

Quantitative / Real-Time PCR

Real-time PCR monitors the amplification of DNA in real time.
It involves using fluorescent dyes that bind to the DNA product, allowing for quantification.

Quantitative / Real-time PCR: SyBr Green

SyBr Green is a fluorescent dye that binds to double-stranded DNA.
Melting curve analysis identifies the melting temperature of DNA sequences to characterize and identify products.

Sanger Sequencing

Sanger sequencing relies on chain termination by dideoxynucleotides.
Fragments of varying lengths are separated by electrophoresis, allowing for DNA sequencing.

Next (Second) Generation Sequencing: Solexa/Illumina Sequencing

This technology allows for high-throughput sequencing by using massive parallel sequencing of DNA fragments.
DNA fragments are fragmented, adapters are attached, and the fragments are sequenced by synthesis.

Second Generation Sequencing: Bridge Amplification

Bridge amplification creates clusters of DNA fragments on the flow cell.
This process amplifies fragments in preparation for sequencing.

Second Generation Sequencing: Cluster Generation

The double stranded molecules are denatured to allow replication of additional copies in preparation for sequencing.
Clonal single molecular arrays are used to produce dense clusters of DNA fragments.

Second Generation Sequencing: Sequencing by Synthesis

Sequencing by synthesis involves sequential incorporation of nucleotides and detection of fluorescence.
Optical sensors and computers are used to determine the order of nucleotides.

Third Generation Sequencing Technologies: Nanopore Sequencing

DNA is fed through a protein pore, and the passage of each base causes a change in current.
This technology allows for long-read sequencing.

DNA-Protein Interaction: Chromatin Immunoprecipitation (ChIP)

Chromatin is crosslinked, and antibodies target specific proteins.
DNA is purified, and the targeted DNA sequence is identified by PCR or sequencing.

DNA-Protein Interaction: Chromatin Immunoprecipitation-seq (ChIP-seq)

Chromatin immunoprecipitation followed by massively parallel sequencing.
This technology identifies DNA-protein interactions genome-wide.

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Description

Test your knowledge on the principles of nucleic acid purification and analysis. This quiz covers topics such as absorbance measurements, the role of RNA, and methods for estimating DNA and RNA concentrations. Challenge yourself with questions on techniques used in the lab and their applications in molecular biology.