COPY: DNA Sequencing: Basics and Significance
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Questions and Answers

What does the movement speed in the gel depend on?

  • Presence of specific nucleotides
  • Fragment composition
  • Fragment length (correct)
  • The column it's found in

    • Where are the smallest DNA fragments located in the gel?

  • In the middle
  • At the bottom (correct)
  • At the top
  • Across all levels

    • How do you determine the sequence of newly synthesized DNA from gel electrophoresis?

  • Reading diagonally
  • Reading from right to left
  • Reading from bottom to top (correct)
  • Reading from top to bottom

    • What do the columns in gel electrophoresis represent?

    <p>Different tubes</p> Signup and view all the answers

    Which end of the DNA strand does the bottom of the gel represent?

    <p>5' end</p> Signup and view all the answers

    How does gel electrophoresis aid in determining the DNA sequence?

    <p>By separating fragments based on size</p> Signup and view all the answers

    What is the main purpose of DNA sequencing?

    <p>Identifying genes and their localization</p> Signup and view all the answers

    How does DNA sequencing assist in predicting protein structure?

    <p>By analyzing the sequence of nucleotides</p> Signup and view all the answers

    What role do bioinformatic tools play in DNA sequencing?

    <p>Aiding in predicting protein structure with high accuracy</p> Signup and view all the answers

    How can DNA sequencing help in identifying disease-susceptibility?

    <p>By determining genetic variations and mutations</p> Signup and view all the answers

    In DNA sequencing, what are codons responsible for?

    <p>Amino acid sequence determination</p> Signup and view all the answers

    Why is comparing unknown DNA sequences to a normal human genome database important?

    <p>To identify genetic variations and disease-causing mutations</p> Signup and view all the answers

    What is the difference between a polymorphism and a mutation based on population frequency?

    <p>A polymorphism exists in more than 1% of the population, while a mutation exists in less than 1%.</p> Signup and view all the answers

    If a DNA sequence shows a band at the same level in columns A and T, what does this indicate?

    <p>Mutation or polymorphism in both chromosomes (homozygous).</p> Signup and view all the answers

    How would an individual have a band at different levels in columns A and T?

    <p>Mutation or polymorphism in one chromosome (heterozygous).</p> Signup and view all the answers

    What does it mean when a DNA sequenced individual has dATP at nucleotide 10 in one chromosome and dTTP at nucleotide 10 in the other?

    <p>Mutation or polymorphism in one chromosome (heterozygous).</p> Signup and view all the answers

    How can we determine if a genetic variant or mutation is pathogenic or not?

    <p>By using fluorescence-based DNA sequencing.</p> Signup and view all the answers

    Why is working with radioactivity considered hazardous?

    <p>It causes health risks and safety concerns.</p> Signup and view all the answers

    What is the main purpose of determining the sequence of genomes of simple organisms like viruses and bacteria first?

    <p>Because they are small and easy to handle</p> Signup and view all the answers

    Why was the mitochondrial DNA sequenced after viral and bacterial genomes?

    <p>Because it was easier to handle than other eukaryotic genomes</p> Signup and view all the answers

    When was the determination of the base sequence in the human genome initiated?

    <p>In 1990</p> Signup and view all the answers

    What is the purpose of using dideoxyribonucleotides in DNA sequencing?

    <p>To prevent further nucleotide addition in DNA strands</p> Signup and view all the answers

    Why is a primer necessary for the initiation of DNA synthesis?

    <p>Because DNA polymerase cannot start synthesis from scratch</p> Signup and view all the answers

    What is the function of the primer in the DNA sequencing process?

    <p>To label incoming nucleotides for attachment</p> Signup and view all the answers

    Why do we add a dideoxynucleotide to the developing DNA strand during sequencing?

    <p>To prevent formation of phosphodiester bonds</p> Signup and view all the answers

    'ddATP' and 'ddCTP' are examples of which type of nucleotides used in DNA sequencing?

    <p>'ddATP' and 'ddCTP' are dideoxyribonucleoside triphosphates</p> Signup and view all the answers

    What do deoxyribonucleotides lack compared to dideoxyribonucleotides?

    <p>Hydroxyl group on carbon 3</p> Signup and view all the answers

    'Phosphodiester bonds form between which groups in the process of DNA synthesis?'

    <p>'OH' group on carbon 3 and phosphate on carbon 5</p> Signup and view all the answers

    What is the purpose of adding ddNTPs to the DNA sequencing reaction?

    <p>To stop the synthesis at specific nucleotides</p> Signup and view all the answers

    How are DNA fragments separated in the gel despite having a difference of just one nucleotide?

    <p>Based on their size and high-resolution gel electrophoresis</p> Signup and view all the answers

    What colors do DNA fragments ending with ddCTP and ddTTP fluoresce with, respectively?

    <p>Blue and yellow</p> Signup and view all the answers

    What determines the color of the peaks obtained in DNA sequencing results?

    <p>The type of ddNTP added</p> Signup and view all the answers

    How does the instrument used for reading DNA sequences translate colors into letters?

    <p>By transforming each color into a corresponding letter</p> Signup and view all the answers

    Why is it necessary to have a high-resolution gel for separating DNA fragments that differ by one nucleotide?

    <p>To distinguish fragments based on small size differences</p> Signup and view all the answers

    What did scientists suggest using instead of radioactive phosphorus for labeling the primer in DNA sequencing?

    <p>Fluorescence</p> Signup and view all the answers

    Why did scientists recommend automating the DNA sequencing process?

    <p>To reduce laborious manual steps</p> Signup and view all the answers

    What additional fluorescent nucleotide is used in DNA sequencing alongside dATP, dTTP, dGTP, and dCTP?

    <p>ddATP (red fluorescence)</p> Signup and view all the answers

    What unique feature do dideoxynucleotides (ddNTP) possess in the DNA sequencing reaction?

    <p>Termination of DNA strand elongation</p> Signup and view all the answers

    Why are fluorescent substrates like ddCTP and ddGTP used in DNA sequencing reactions?

    <p>To allow visualization of sequence termination points</p> Signup and view all the answers

    If a DNA sequence shows a band at the same level in columns C and G, what does this indicate?

    <p>The DNA strands are anti-parallel</p> Signup and view all the answers

    What does it mean when two bands appear at the same level in a DNA gel electrophoresis?

    <p>They have the same size</p> Signup and view all the answers

    Why do we need to switch the gel in hypothetical DNA sequencing?

    <p>To visualize the template sequence</p> Signup and view all the answers

    In DNA sequencing, why is it important to remember that DNA is anti-parallel?

    <p>To ensure correct base pairing</p> Signup and view all the answers

    What is the significance of complementary strands of DNA in determining template sequences?

    <p>They provide a guide for determining template sequences</p> Signup and view all the answers

    Why is it unnecessary to rely on gel electrophoresis to find template sequences in DNA sequencing?

    <p>The template can be easily identified by flipping the sequences</p> Signup and view all the answers

    What does the camera detect in the DNA sequencing process?

    <p>The color of the added nucleotides in each cluster</p> Signup and view all the answers

    What is the final DNA sequence in cluster 3 based on the provided text?

    <p>It is not mentioned</p> Signup and view all the answers

    How do bioinformatics tools contribute to DNA sequencing?

    <p>By combining information to determine the DNA sequence</p> Signup and view all the answers

    What happens during the second round of nucleotide addition in DNA sequencing?

    <p>Multiple nucleotides are activated for addition</p> Signup and view all the answers

    Why are different colors generated from each cluster in DNA sequencing?

    <p>To represent the added nucleotides</p> Signup and view all the answers

    How is the DNA sequence of all clusters determined in DNA sequencing?

    <p>By combining information from all clusters</p> Signup and view all the answers

    What was the first mitochondrial DNA sequenced after?

    <p>Viral genomes</p> Signup and view all the answers

    Why was the yeast genome sequenced before the human genome?

    <p>Yeast is easier to handle</p> Signup and view all the answers

    Which genome was among the first eukaryotic genomes to be sequenced?

    <p>Yeast genome</p> Signup and view all the answers

    In DNA synthesis, where is the phosphodiester bond formed?

    <p>Between the hydroxyl group and the phosphate group</p> Signup and view all the answers

    What is the purpose of a primer in DNA synthesis?

    <p>To initiate DNA synthesis</p> Signup and view all the answers

    Why did DNA sequencing start with viruses and prokaryotes?

    <p>Because they have fewer chromosomes</p> Signup and view all the answers

    Which nucleotide type is used to stop DNA synthesis in sequencing?

    <p>'ddCTP'</p> Signup and view all the answers

    What does a Dideoxyribonucleotide lack compared to a deoxyribonucleotide?

    <p>Hydroxyl groups on carbons 2 and 3</p> Signup and view all the answers

    'Mbp' in genetics stands for what measurement?

    <p>'Mega base pairs'</p> Signup and view all the answers

    What is the substrate of DNA polymerase in DNA synthesis?

    <p>Deoxynucleotides (A,T,C,G)</p> Signup and view all the answers

    What is the purpose of incorporating a dideoxynucleotide in each reaction during DNA sequencing?

    <p>To inhibit further DNA synthesis by stopping nucleotide addition</p> Signup and view all the answers

    Why does DNA synthesis terminate when a dideoxynucleotide is added by the DNA polymerase?

    <p>The dideoxynucleotide lacks a 3' hydroxyl group for nucleotide addition</p> Signup and view all the answers

    How are different lengths of DNA fragments generated during the Sanger sequencing method?

    <p>By the random termination of DNA synthesis by ddNTPs</p> Signup and view all the answers

    Why is gel electrophoresis used after DNA sequencing reactions?

    <p>To visualize fragments based on size</p> Signup and view all the answers

    What role does the labeled primer play in the Sanger sequencing method?

    <p>Determines the template DNA's sequence order</p> Signup and view all the answers

    How does the addition of a dideoxynucleotide affect DNA polymerase activity during sequencing?

    <p>Inhibits further nucleotide addition leading to termination</p> Signup and view all the answers

    Why are different lengths of labeled DNA molecules generated during Sanger sequencing?

    <p>Because of random termination by dideoxynucleotides</p> Signup and view all the answers

    What happens when a dideoxyribonucleotide is mistakenly added instead of a deoxyribonucleotide during sequencing?

    <p>Results in premature termination of DNA synthesis</p> Signup and view all the answers

    Why do different fragments of DNA have varying lengths in Sanger sequencing?

    <p>As a result of random termination by dideoxynucleotides</p> Signup and view all the answers

    What is the significance of separating DNA fragments by size using gel electrophoresis after Sanger sequencing?

    <p>To visualize and order fragments for sequence determination</p> Signup and view all the answers

    What is the significance of having ddATP along with dATP in tube 1 during DNA synthesis?

    <p>It allows for the rapid termination of some DNA fragments.</p> Signup and view all the answers

    What is the consequence of adding a dideoxynucleotide to a DNA strand during synthesis?

    <p>It causes the termination of DNA synthesis at that point.</p> Signup and view all the answers

    Why do DNA fragments synthesized in tube 3 end with ddCTP?

    <p>As a result of ddCTP causing premature termination of synthesis.</p> Signup and view all the answers

    What distinguishes the resulting DNA fragments in tube 2 from those in tube 1?

    <p>Fragments in tube 2 end with thymine.</p> Signup and view all the answers

    What determines the different lengths of DNA fragments produced during Sanger sequencing?

    <p>The presence of dideoxynucleotides leading to premature termination.</p> Signup and view all the answers

    Why is it important to label the primer used in Sanger sequencing?

    <p>To indicate the beginning point for DNA synthesis.</p> Signup and view all the answers

    What was the purpose of sequencing the genome of simple organisms like viruses and bacteria first?

    <p>To start with smaller and easier-to-handle genomes</p> Signup and view all the answers

    Where does the phosphodiester bond form during DNA synthesis?

    <p>Between the hydroxyl group of one nucleotide and the phosphate of another</p> Signup and view all the answers

    What role does the labeled primer play in DNA sequencing?

    <p>It acts as a starting point for DNA synthesis</p> Signup and view all the answers

    Why was yeast genome sequenced before the human genome?

    <p>To understand simpler eukaryotic genomes first</p> Signup and view all the answers

    How does DNA synthesis terminate when a dideoxynucleotide is added by the DNA polymerase?

    <p>A phosphodiester bond cannot form</p> Signup and view all the answers

    What is the significance of determining the base sequence in the human genome?

    <p>To comprehend cellular functions and diseases in humans</p> Signup and view all the answers

    'ddATP' and 'ddCTP' are examples of which type of nucleotides used in DNA sequencing?

    <p>'Terminating' nucleotides that halt DNA synthesis</p> Signup and view all the answers

    What do dideoxynucleotides lack compared to deoxyribonucleotides?

    <p>They lack a hydroxyl group on carbon number 3</p> Signup and view all the answers

    What was the basic method of DNA sequencing based on originally?

    <p>Employing di-deoxyribonucleotides</p> Signup and view all the answers

    What information can be deduced when an individual has two overlapping bands on the DNA sequencing diagram?

    <p>The individual is heterozygous and has a polymorphism.</p> Signup and view all the answers

    How is an individual determined to be homozygous based on DNA sequencing results?

    <p>By detecting two peaks at the same location for a nucleotide different from the expected one.</p> Signup and view all the answers

    What is the significance of having two peaks on the DNA sequencing diagram for an individual?

    <p>It indicates homozygosity with polymorphism.</p> Signup and view all the answers

    How does DNA sequencing reveal if an individual is homozygous for a mutated gene?

    <p>By displaying two peaks at a nucleotide location where there should be one.</p> Signup and view all the answers

    What does it suggest when a DNA sequencing diagram displays two peaks at the same position for an individual?

    <p>The individual has two different alleles for the same gene.</p> Signup and view all the answers

    How is a homozygous mutation distinguished from a heterozygous polymorphism in DNA sequencing results?

    <p>By identifying overlapping bands at specific locations.</p> Signup and view all the answers

    What does it indicate when two bands appear at the same level in columns A and T?

    <p>The person has a mutation in both chromosomes</p> Signup and view all the answers

    What does it mean if a person is homozygous for a specific nucleotide sequence at a certain level?

    <p>The person has a mutation in both chromosomes</p> Signup and view all the answers

    In DNA sequencing, what does it indicate when two individuals have the same nucleotide at a specific position on both chromosomes?

    <p>Both individuals have a mutation in one chromosome</p> Signup and view all the answers

    If an individual shows a band at different levels in columns A and T, what does this suggest?

    <p>The individual has a polymorphism in one chromosome</p> Signup and view all the answers

    What does it mean when a person has two bands at different levels in columns A and T?

    <p>The person is heterozygous for the mutation</p> Signup and view all the answers

    In DNA sequencing, what does it suggest if an individual has different nucleotides on their maternal and paternal chromosomes at the same position?

    <p>The individual has a polymorphism in one chromosome</p> Signup and view all the answers

    What is the main reason that incorporation of a dideoxynucleotide stops further DNA synthesis?

    <p>It lacks a 3' hydroxyl group for the addition of the next nucleotide</p> Signup and view all the answers

    What is the role of a primer in the Sanger sequencing method?

    <p>To initiate DNA synthesis at a known sequence</p> Signup and view all the answers

    In Sanger sequencing, why does the addition of a dideoxynucleotide terminate DNA synthesis?

    <p>It prevents elongation due to lack of a 3' hydroxyl group</p> Signup and view all the answers

    How are different lengths of DNA fragments generated in Sanger sequencing?

    <p>By stopping synthesis at different points using dideoxynucleotides</p> Signup and view all the answers

    What determines the size of each DNA fragment in Sanger sequencing?

    <p>The terminal dideoxynucleotide added to the growing chain</p> Signup and view all the answers

    Why is gel electrophoresis used after Sanger sequencing reactions?

    <p>To separate DNA fragments based on size</p> Signup and view all the answers

    What is the significance of labeling the primer with radioactive phosphorus in DNA sequencing?

    <p>To visualize the attached fragment on X-ray film</p> Signup and view all the answers

    How does the Sanger sequencing method determine the order of nucleotides in a DNA sequence?

    <p>By reading the terminal dideoxynucleotides of separated fragments</p> Signup and view all the answers

    How does gel electrophoresis aid in interpreting DNA sequences?

    <p>By separating fragments based on size for visualization</p> Signup and view all the answers

    What does each labeled DNA molecule represent in Sanger sequencing?

    <p>A specific nucleotide sequence</p> Signup and view all the answers

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