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?

Small physical scale on a microscope Signup and view all the answers

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

<blockquote> 10,000 bp </blockquote> Signup and view all the answers

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

To break apart DNA or RNA backbones Signup and view all the answers

Which statement accurately describes polymerases?

They operate by utilizing nucleotide 5-triphosphates. Signup and view all the answers

What characteristic distinguishes restriction endonucleases from other enzymes?

They cleave DNA at specific recognition sequences. Signup and view all the answers

How does RNA self-cleavage occur?

With the assistance of metal cations and heat. Signup and view all the answers

What is the final product of DNA polymerase function?

A nucleic acid polymer extending from a template. Signup and view all the answers

Which process is involved in making and breaking phosphoester bonds?

Transesterification reactions. Signup and view all the answers

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

New nucleotides are always added to the 3' end of the strand. Signup and view all the answers

What role does pyrophosphate play during nucleic acid synthesis?

It is a byproduct that improves thermodynamics. Signup and view all the answers

What distinguishes exonucleases from endonucleases?

Exonucleases cut at the ends of nucleic acid strands. Signup and view all the answers

What is one method of DNA sequencing discussed?

Sanger sequencing. Signup and view all the answers

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

Because of spontaneous hydrolysis occurring over time. Signup and view all the answers

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

Formation of a phosphate bond between two distinct strands. Signup and view all the answers

What is a common method that improves DNA sequencing bandwidth?

Using long reads of 10,000+ base pairs. Signup and view all the answers

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

Covalent bonds Signup and view all the answers

Which of the following best describes deamination?

The loss of an exocyclic amino group Signup and view all the answers

What is the typical consequence of cytosine deamination in DNA?

Leads to uracil formation from cytosine Signup and view all the answers

Why is thymine preferred over uracil in DNA?

Thymine has a methyl group which helps distinguish it from uracil Signup and view all the answers

What is the role of APOBEC enzymes in cellular function?

They edit mRNA via deamination of cytosine Signup and view all the answers

How does methylation of cytosine affect gene expression?

It often represses gene expression Signup and view all the answers

What process allows the identification of ancient DNA through deamination?

High frequency C→U changes over time Signup and view all the answers

What is the main effect of enzymatic adenine deamination?

Conversion to hypoxanthine Signup and view all the answers

What distinguishes bacterial from eukaryotic methylation of cytosine?

Only eukaryotes methylate at CpG sites Signup and view all the answers

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

Mutations could occur due to pair misalignment Signup and view all the answers

Which bonding change occurs during cytosine methylation?

Adds a methyl group without changing base pairing Signup and view all the answers

What is the spontaneous rate of cytosine deamination per cell?

5 events per day Signup and view all the answers

What type of genetic editing is related to deaminases?

Base editing Signup and view all the answers

What is the relationship between deamination and mutation rates?

Deamination increases mutation rates Signup and view all the answers

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

It serves as the methyl group donor. Signup and view all the answers

What is the role of dideoxynucleotides in Sanger sequencing?

To terminate the DNA chain Signup and view all the answers

Which reagents are typically involved in Sanger sequencing?

dATP, dCTP, dGTP, and ddNTPs Signup and view all the answers

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

dideoxynucleotides Signup and view all the answers

What is a significant advantage of modern Sanger sequencing?

It allows simultaneous termination and labeling of DNA Signup and view all the answers

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

Reversible Terminator Sequencing Signup and view all the answers

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

It generates fewer read lengths Signup and view all the answers

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

Dideoxynucleotides Signup and view all the answers

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

Capillary electrophoresis Signup and view all the answers

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

To create a mixture of fragment lengths Signup and view all the answers

What distinguishes ddNTPs from regular deoxynucleotides in their structure?

A missing hydroxyl group at the 3' position Signup and view all the answers

What type of polymerase is primarily used in Sanger sequencing?

DNA polymerase Signup and view all the answers

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

Automated capillary electrophoresis Signup and view all the answers

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

To provide identification of specific nucleotides Signup and view all the answers

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

They lack a key hydroxyl group Signup and view all the answers

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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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.