Nucleic Acid Chemistry Lecture 2
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Questions and Answers

What is a primary advantage of Single Molecule Sequencing (PacBio)?

  • Higher throughput than other methods
  • Lower physical complexity
  • Reduced cost per sequencing run
  • Ability to read lengths averaging over 10,000 bp (correct)

What is a significant disadvantage of Single Molecule Sequencing?

  • It uses a simpler flow cell design
  • It enables sequencing of very short DNA fragments
  • It provides a higher number of reads compared to traditional methods
  • Its physical complexity results in lower throughput (correct)

What is the estimated read count possible with Single Molecule Sequencing?

  • About 1 million reads
  • Around 10 million reads (correct)
  • Over 1 billion reads
  • Approximately 100 million reads

What factor primarily allows Illumina sequencing to have high bandwidth?

<p>Small physical scale on a microscope (B)</p> Signup and view all the answers

How many base pairs does Single Molecule Sequencing typically average read lengths of?

<blockquote> <p>10,000 bp (D)</p> </blockquote> Signup and view all the answers

What is the primary role of nucleases in nucleic acid chemistry?

<p>To break apart DNA or RNA backbones (C)</p> Signup and view all the answers

Which statement accurately describes polymerases?

<p>They operate by utilizing nucleotide 5-triphosphates. (C)</p> Signup and view all the answers

What characteristic distinguishes restriction endonucleases from other enzymes?

<p>They cleave DNA at specific recognition sequences. (A)</p> Signup and view all the answers

How does RNA self-cleavage occur?

<p>With the assistance of metal cations and heat. (A)</p> Signup and view all the answers

What is the final product of DNA polymerase function?

<p>A nucleic acid polymer extending from a template. (A)</p> Signup and view all the answers

Which process is involved in making and breaking phosphoester bonds?

<p>Transesterification reactions. (D)</p> Signup and view all the answers

What defines the direction of nucleic acid polymer synthesis by polymerases?

<p>New nucleotides are always added to the 3' end of the strand. (A)</p> Signup and view all the answers

What role does pyrophosphate play during nucleic acid synthesis?

<p>It is a byproduct that improves thermodynamics. (A)</p> Signup and view all the answers

What distinguishes exonucleases from endonucleases?

<p>Exonucleases cut at the ends of nucleic acid strands. (B)</p> Signup and view all the answers

What is one method of DNA sequencing discussed?

<p>Sanger sequencing. (C)</p> Signup and view all the answers

Why do nucleic acids have a half-life of about 4 years at neutral pH and 25ºC?

<p>Because of spontaneous hydrolysis occurring over time. (B)</p> Signup and view all the answers

What initiates the reaction catalyzed by ligases in nucleic acid processing?

<p>Formation of a phosphate bond between two distinct strands. (A)</p> Signup and view all the answers

What is a common method that improves DNA sequencing bandwidth?

<p>Using long reads of 10,000+ base pairs. (B)</p> Signup and view all the answers

What type of bonding is primarily involved in the making and breaking of phosphoester bonds in nucleic acids?

<p>Covalent bonds (A)</p> Signup and view all the answers

Which of the following best describes deamination?

<p>The loss of an exocyclic amino group (B)</p> Signup and view all the answers

What is the typical consequence of cytosine deamination in DNA?

<p>Leads to uracil formation from cytosine (A)</p> Signup and view all the answers

Why is thymine preferred over uracil in DNA?

<p>Thymine has a methyl group which helps distinguish it from uracil (B)</p> Signup and view all the answers

What is the role of APOBEC enzymes in cellular function?

<p>They edit mRNA via deamination of cytosine (A)</p> Signup and view all the answers

How does methylation of cytosine affect gene expression?

<p>It often represses gene expression (B)</p> Signup and view all the answers

What process allows the identification of ancient DNA through deamination?

<p>High frequency C→U changes over time (A)</p> Signup and view all the answers

What is the main effect of enzymatic adenine deamination?

<p>Conversion to hypoxanthine (A)</p> Signup and view all the answers

What distinguishes bacterial from eukaryotic methylation of cytosine?

<p>Only eukaryotes methylate at CpG sites (A)</p> Signup and view all the answers

What would happen if uracil is not efficiently repaired in DNA?

<p>Mutations could occur due to pair misalignment (B)</p> Signup and view all the answers

Which bonding change occurs during cytosine methylation?

<p>Adds a methyl group without changing base pairing (C)</p> Signup and view all the answers

What is the spontaneous rate of cytosine deamination per cell?

<p>5 events per day (A)</p> Signup and view all the answers

What type of genetic editing is related to deaminases?

<p>Base editing (A)</p> Signup and view all the answers

What is the relationship between deamination and mutation rates?

<p>Deamination increases mutation rates (D)</p> Signup and view all the answers

What is one of the roles of S-adenosylmethionine in methylation reactions?

<p>It serves as the methyl group donor. (C)</p> Signup and view all the answers

What is the role of dideoxynucleotides in Sanger sequencing?

<p>To terminate the DNA chain (B)</p> Signup and view all the answers

Which reagents are typically involved in Sanger sequencing?

<p>dATP, dCTP, dGTP, and ddNTPs (B)</p> Signup and view all the answers

In the context of Sanger sequencing, the term 'chain terminator' refers to which component?

<p>dideoxynucleotides (C)</p> Signup and view all the answers

What is a significant advantage of modern Sanger sequencing?

<p>It allows simultaneous termination and labeling of DNA (B)</p> Signup and view all the answers

What technology is mentioned as an advancement over traditional Sanger sequencing?

<p>Reversible Terminator Sequencing (A)</p> Signup and view all the answers

What is one disadvantage of Reversible Terminator Sequencing compared to Sanger sequencing?

<p>It generates fewer read lengths (D)</p> Signup and view all the answers

Which component prevents additional nucleophilic attacks from occurring during DNA synthesis in Sanger sequencing?

<p>Dideoxynucleotides (B)</p> Signup and view all the answers

What process is used to visualize the results of Sanger sequencing?

<p>Capillary electrophoresis (C)</p> Signup and view all the answers

Why is a small amount of dideoxynucleotide triphosphates (ddNTPs) added in Sanger sequencing reactions?

<p>To create a mixture of fragment lengths (C)</p> Signup and view all the answers

What distinguishes ddNTPs from regular deoxynucleotides in their structure?

<p>A missing hydroxyl group at the 3' position (B)</p> Signup and view all the answers

What type of polymerase is primarily used in Sanger sequencing?

<p>DNA polymerase (C)</p> Signup and view all the answers

Which feature of Sanger sequencing permits the analysis of DNA sequences?

<p>Automated capillary electrophoresis (B)</p> Signup and view all the answers

The use of fluorescent labels in modern Sanger sequencing primarily serves what purpose?

<p>To provide identification of specific nucleotides (C)</p> Signup and view all the answers

What characteristic of ddNTPs makes them suitable for chain termination in Sanger sequencing?

<p>They lack a key hydroxyl group (D)</p> Signup and view all the answers

Flashcards

Spontaneous cytosine deamination

A natural chemical reaction where cytosine converts to uracil.

Cytosine to uracil mutation

The change in DNA sequence from cytosine to uracil due to deamination.

Deamination of cytosine

Loss of an amino group from cytosine, producing uracil.

Thymine in DNA

DNA base that is chemically distinct , to prevent confusion with uracil.

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DNA repair

Cellular mechanisms to fix damages to DNA.

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5-methylcytosine

A methylated form of cytosine, similar to thymine in its base pairing.

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Cytosine methylation

Adding a methyl group to cytosine, often at CpG sites.

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CpG sites

Areas in DNA where cytosine is followed by guanine.

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Adenine deamination

Adenine changing to hypoxanthine.

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Activation-induced cytidine deaminase (AID)

Enzyme that alters antibody genes to create diversity.

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APOBEC enzymes

Enzymes that edit mRNA, sometimes to counter viruses.

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Base editing

A kind of genome editing that chemically changes single bases.

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Ancient DNA sequencing

Process of reading genetic material from preserved remains.

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Inosine

The nucleoside of hypoxanthine. Important for mRNA editing.

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Bacterial cytosine methylation

A way bacterial DNA differentiates itself from viruses.

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DNA sequencing methods

Techniques used to determine the order of nucleotides in a DNA molecule.

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Illumina sequencing

Sequencing method that sequences DNA molecules in small clusters on a flow cell.

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Single Molecule Sequencing (SMRT)

Sequencing method that sequences DNA molecules one at a time enabling longer reads.

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PacBio sequencing

A type of Single Molecule Sequencing (SMRT).

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Read length

The average length of DNA sequences determined in a single sequencing run.

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Transesterification

A chemical reaction where a bond is broken and remade in a nucleic acid backbone, involving a nucleophilic attack.

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Nucleic Acid Backbone

The repeating sugar-phosphate-base units that form the structural framework of nucleic acids.

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Nucleophile

An atom or molecule that is attracted to and donates electrons to an electron-deficient center.

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Nuclease

An enzyme that breaks down the nucleic acid backbone.

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Exonuclease

A nuclease that cuts nucleic acids from the ends.

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Endonuclease

A nuclease that cuts nucleic acids in the middle.

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Polymerase

An enzyme that builds nucleic acids by adding nucleotides to an existing strand.

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Ligase

An enzyme that joins two nucleic acid fragments together.

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Restriction Endonuclease

An endonuclease that cuts DNA at specific sequences.

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dNTP

Deoxynucleotide triphosphate. Building block for DNA synthesis.

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NTP

Nucleotide triphosphate. Building block for RNA synthesis.

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3' end

The end of a nucleic acid strand with a free 3' hydroxyl group.

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5' end

The end of a nucleic acid strand with a free 5' phosphate group.

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RNA self-cleavage

RNA can cut itself, a special reaction.

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DNA sequencing

Methods for determining the order of nucleotides in a DNA molecule.

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Dideoxynucleotides (ddNTPs)

Modified nucleotides that lack a 3'-hydroxyl group, halting DNA synthesis during sequencing.

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Sanger Sequencing

A method for determining DNA sequences using dideoxynucleotides (ddNTPs).

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Chain Termination

The process of stopping DNA synthesis by incorporating a ddNTP.

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Gel Electrophoresis

A method to separate DNA fragments based on size.

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Fluorescent ddNTPs

Dideoxynucleotides labeled with fluorescent tags for DNA sequencing.

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Capillary Electrophoresis

A method to separate DNA fragments during sequencing using capillary tubes.

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Reversible Terminators

Modified nucleotides used in Illumina sequencing, allowing for multiple cycles.

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DNA Polymerase

Enzyme that synthesizes DNA strands during sequencing.

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ddNTP Incorporation

The process of adding a ddNTP to a growing DNA chain.

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DideoxyTTP

A modified thymidine that terminates DNA synthesis.

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High-Throughput Sequencing

Sequencing that can process millions to billions of DNA molecules simultaneously.

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DNA Fragment

A segment of DNA that is a result of stopping the DNA synthesis reaction.

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Study Notes

Lecture 2: Nucleic Acid Chemistry

  • Nucleic acids undergo chemical reactions
  • These reactions impact the transmission of genetic information
  • Methods for determining the sequence of a DNA molecule are discussed

Common Chemical Reactions

  • Making and breaking phosphoester bonds are common reactions
  • This is a central chemistry for changing nucleic acid backbones

Information Carrier

  • DNA and RNA molecules have a physical layer that allows them to partake in chemical reactions
  • DNA and RNA have specific structures and components (e.g., phosphate, sugar, base)
  • Different bases (Adenine, Guanine, Cytosine, Thymine/Uracil) create specific interactions with each other
  • Phosphodiester linkages connect nucleotides in a strand

Deamination

  • Deamination is a common spontaneous reaction
  • The loss of exocyclic amino groups is called deamination
  • One example of deamination is cytosine converting to uracil
  • ~100 events of cytosine deamination occur per day in a cell
  • DNA repair mechanisms recognize uracil as foreign and remove it
  • Thymine replaces uracil in DNA; this most likely is due to repair to distinguish normal bases from damaged ones

Deamination & Cytosine Mutations

  • Spontaneous cytosine deamination leads to mutations
  • The rate of cytosine deamination is approximately 5 x 10⁻¹³ s⁻¹ (~1 in 50,000 years)
  • Approximately 3 billion cytosines exist in a cell
  • C→U changes in DNA replication can lead to mutations that change C to U and G to A

Deamination and Thymine vs Uracil

  • Thymine is methylated cytosine. The methyl group distinguishes thymine from uracil. This helps the cell effectively remove uracil.

Ancient DNA Highlight

  • Ancient DNA sequencing relies on the spontaneous cytosine deamination to uracil

Enzymatic Cytosine Deamination

  • Activation-induced deaminase (AID) participates in antibody gene mutation in B lymphocytes
  • APOBEC enzymes are responsible for an editing mechanism that leads to C→U changes in some genes, affecting protein coding

Adenine Deamination

  • Adenine can be deaminated to hypoxanthine (or inosine)
  • This reaction is responsible for a type of editing mechanism in mRNA

Cytosine Methylation

  • Cytosine methylation is analogous to thymine
  • No changes occur to H-bonding or base pairing
  • The methyl group differentiates 5-methylcytosine

Methylation and Deamination Summary

  • Cytosine can be methylated and deaminated
  • Enzymes involved: S-adenosylmethionine and AID, APOBEC
  • Methylation in bacteria differentiates self-DNA from viral/bacteriophage DNA
  • Eukaryotic methylation occurs at CpG sites; affects gene expression
  • DNA repair mechanisms remove uracil formed by cytosine deamination

Enzymes

  • Nucleases break down DNA/RNA backbones during hydrolysis (transesterification onto water). There are exonucleases and endonucleases, with differences in their actions.
  • Polymerases join nucleotides together into polymers of nucleic acid. They typically add to the 3' end of DNA or RNA.
  • Ligases connect the 3' end of one polymer to the 5' end of another polymer

Restriction Endonucleases

  • Restriction endonucleases are enzymes that cut DNA at specific sequences.
  • Bacteria use them to defend against viruses
  • Sequences recognized by restriction endonucleases are usually 4-8 base pairs long

Polymerases Synthesizing Nucleic Acids

  • Polymerases catalyze the addition of nucleotides (NTPs/dNTPs), using 3'-OH as the nucleophile, to existing DNA or RNA chains
  • Inorganic pyrophosphate is released as a byproduct.

Polymerases In Detail

  • DNA/RNA polymerases use a template strand to synthesize complementary strands. Their 3' end grows by extending 5' to 3'
  • Base pairing of dNTPs with the template is important in this process

DNA Sequencing Methods

  • Sanger sequencing uses dideoxynucleotides
  • Illumina sequencing utilizes a reversible terminator method
  • PacBio utilizes single-molecule sequencing technology.

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MCB102 Fall 2024 Lecture 2: Nucleic Acid Chemistry PDF

Description

Dive into the fascinating world of nucleic acid chemistry with this quiz covering essential reactions and structures. Explore how these reactions influence genetic information transmission and learn about critical processes like deamination. Test your understanding of DNA and RNA's chemical properties and their components.

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