108 Questions
What does the movement speed in the gel depend on?
Fragment length
Where are the smallest DNA fragments located in the gel?
At the bottom
How do you determine the sequence of newly synthesized DNA from gel electrophoresis?
Reading from bottom to top
What do the columns in gel electrophoresis represent?
Different tubes
Which end of the DNA strand does the bottom of the gel represent?
5' end
How does gel electrophoresis aid in determining the DNA sequence?
By separating fragments based on size
What is the main purpose of DNA sequencing?
Identifying genes and their localization
How does DNA sequencing assist in predicting protein structure?
By analyzing the sequence of nucleotides
What role do bioinformatic tools play in DNA sequencing?
Aiding in predicting protein structure with high accuracy
How can DNA sequencing help in identifying disease-susceptibility?
By determining genetic variations and mutations
In DNA sequencing, what are codons responsible for?
Amino acid sequence determination
Why is comparing unknown DNA sequences to a normal human genome database important?
To identify genetic variations and disease-causing mutations
What is the difference between a polymorphism and a mutation based on population frequency?
A polymorphism exists in more than 1% of the population, while a mutation exists in less than 1%.
If a DNA sequence shows a band at the same level in columns A and T, what does this indicate?
Mutation or polymorphism in both chromosomes (homozygous).
How would an individual have a band at different levels in columns A and T?
Mutation or polymorphism in one chromosome (heterozygous).
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?
Mutation or polymorphism in one chromosome (heterozygous).
How can we determine if a genetic variant or mutation is pathogenic or not?
By using fluorescence-based DNA sequencing.
Why is working with radioactivity considered hazardous?
It causes health risks and safety concerns.
What is the main purpose of determining the sequence of genomes of simple organisms like viruses and bacteria first?
Because they are small and easy to handle
Why was the mitochondrial DNA sequenced after viral and bacterial genomes?
Because it was easier to handle than other eukaryotic genomes
When was the determination of the base sequence in the human genome initiated?
In 1990
What is the purpose of using dideoxyribonucleotides in DNA sequencing?
To prevent further nucleotide addition in DNA strands
Why is a primer necessary for the initiation of DNA synthesis?
Because DNA polymerase cannot start synthesis from scratch
What is the function of the primer in the DNA sequencing process?
To label incoming nucleotides for attachment
Why do we add a dideoxynucleotide to the developing DNA strand during sequencing?
To prevent formation of phosphodiester bonds
'ddATP' and 'ddCTP' are examples of which type of nucleotides used in DNA sequencing?
'ddATP' and 'ddCTP' are dideoxyribonucleoside triphosphates
What do deoxyribonucleotides lack compared to dideoxyribonucleotides?
Hydroxyl group on carbon 3
'Phosphodiester bonds form between which groups in the process of DNA synthesis?'
'OH' group on carbon 3 and phosphate on carbon 5
What is the purpose of adding ddNTPs to the DNA sequencing reaction?
To stop the synthesis at specific nucleotides
How are DNA fragments separated in the gel despite having a difference of just one nucleotide?
Based on their size and high-resolution gel electrophoresis
What colors do DNA fragments ending with ddCTP and ddTTP fluoresce with, respectively?
Blue and yellow
What determines the color of the peaks obtained in DNA sequencing results?
The type of ddNTP added
How does the instrument used for reading DNA sequences translate colors into letters?
By transforming each color into a corresponding letter
Why is it necessary to have a high-resolution gel for separating DNA fragments that differ by one nucleotide?
To distinguish fragments based on small size differences
What did scientists suggest using instead of radioactive phosphorus for labeling the primer in DNA sequencing?
Fluorescence
Why did scientists recommend automating the DNA sequencing process?
To reduce laborious manual steps
What additional fluorescent nucleotide is used in DNA sequencing alongside dATP, dTTP, dGTP, and dCTP?
ddATP (red fluorescence)
What unique feature do dideoxynucleotides (ddNTP) possess in the DNA sequencing reaction?
Termination of DNA strand elongation
Why are fluorescent substrates like ddCTP and ddGTP used in DNA sequencing reactions?
To allow visualization of sequence termination points
If a DNA sequence shows a band at the same level in columns C and G, what does this indicate?
The DNA strands are anti-parallel
What does it mean when two bands appear at the same level in a DNA gel electrophoresis?
They have the same size
Why do we need to switch the gel in hypothetical DNA sequencing?
To visualize the template sequence
In DNA sequencing, why is it important to remember that DNA is anti-parallel?
To ensure correct base pairing
What is the significance of complementary strands of DNA in determining template sequences?
They provide a guide for determining template sequences
Why is it unnecessary to rely on gel electrophoresis to find template sequences in DNA sequencing?
The template can be easily identified by flipping the sequences
What does the camera detect in the DNA sequencing process?
The color of the added nucleotides in each cluster
What is the final DNA sequence in cluster 3 based on the provided text?
It is not mentioned
How do bioinformatics tools contribute to DNA sequencing?
By combining information to determine the DNA sequence
What happens during the second round of nucleotide addition in DNA sequencing?
Multiple nucleotides are activated for addition
Why are different colors generated from each cluster in DNA sequencing?
To represent the added nucleotides
How is the DNA sequence of all clusters determined in DNA sequencing?
By combining information from all clusters
What was the first mitochondrial DNA sequenced after?
Viral genomes
Why was the yeast genome sequenced before the human genome?
Yeast is easier to handle
Which genome was among the first eukaryotic genomes to be sequenced?
Yeast genome
In DNA synthesis, where is the phosphodiester bond formed?
Between the hydroxyl group and the phosphate group
What is the purpose of a primer in DNA synthesis?
To initiate DNA synthesis
Why did DNA sequencing start with viruses and prokaryotes?
Because they have fewer chromosomes
Which nucleotide type is used to stop DNA synthesis in sequencing?
'ddCTP'
What does a Dideoxyribonucleotide lack compared to a deoxyribonucleotide?
Hydroxyl groups on carbons 2 and 3
'Mbp' in genetics stands for what measurement?
'Mega base pairs'
What is the substrate of DNA polymerase in DNA synthesis?
Deoxynucleotides (A,T,C,G)
What is the purpose of incorporating a dideoxynucleotide in each reaction during DNA sequencing?
To inhibit further DNA synthesis by stopping nucleotide addition
Why does DNA synthesis terminate when a dideoxynucleotide is added by the DNA polymerase?
The dideoxynucleotide lacks a 3' hydroxyl group for nucleotide addition
How are different lengths of DNA fragments generated during the Sanger sequencing method?
By the random termination of DNA synthesis by ddNTPs
Why is gel electrophoresis used after DNA sequencing reactions?
To visualize fragments based on size
What role does the labeled primer play in the Sanger sequencing method?
Determines the template DNA's sequence order
How does the addition of a dideoxynucleotide affect DNA polymerase activity during sequencing?
Inhibits further nucleotide addition leading to termination
Why are different lengths of labeled DNA molecules generated during Sanger sequencing?
Because of random termination by dideoxynucleotides
What happens when a dideoxyribonucleotide is mistakenly added instead of a deoxyribonucleotide during sequencing?
Results in premature termination of DNA synthesis
Why do different fragments of DNA have varying lengths in Sanger sequencing?
As a result of random termination by dideoxynucleotides
What is the significance of separating DNA fragments by size using gel electrophoresis after Sanger sequencing?
To visualize and order fragments for sequence determination
What is the significance of having ddATP along with dATP in tube 1 during DNA synthesis?
It allows for the rapid termination of some DNA fragments.
What is the consequence of adding a dideoxynucleotide to a DNA strand during synthesis?
It causes the termination of DNA synthesis at that point.
Why do DNA fragments synthesized in tube 3 end with ddCTP?
As a result of ddCTP causing premature termination of synthesis.
What distinguishes the resulting DNA fragments in tube 2 from those in tube 1?
Fragments in tube 2 end with thymine.
What determines the different lengths of DNA fragments produced during Sanger sequencing?
The presence of dideoxynucleotides leading to premature termination.
Why is it important to label the primer used in Sanger sequencing?
To indicate the beginning point for DNA synthesis.
What was the purpose of sequencing the genome of simple organisms like viruses and bacteria first?
To start with smaller and easier-to-handle genomes
Where does the phosphodiester bond form during DNA synthesis?
Between the hydroxyl group of one nucleotide and the phosphate of another
What role does the labeled primer play in DNA sequencing?
It acts as a starting point for DNA synthesis
Why was yeast genome sequenced before the human genome?
To understand simpler eukaryotic genomes first
How does DNA synthesis terminate when a dideoxynucleotide is added by the DNA polymerase?
A phosphodiester bond cannot form
What is the significance of determining the base sequence in the human genome?
To comprehend cellular functions and diseases in humans
'ddATP' and 'ddCTP' are examples of which type of nucleotides used in DNA sequencing?
'Terminating' nucleotides that halt DNA synthesis
What do dideoxynucleotides lack compared to deoxyribonucleotides?
They lack a hydroxyl group on carbon number 3
What was the basic method of DNA sequencing based on originally?
Employing di-deoxyribonucleotides
What information can be deduced when an individual has two overlapping bands on the DNA sequencing diagram?
The individual is heterozygous and has a polymorphism.
How is an individual determined to be homozygous based on DNA sequencing results?
By detecting two peaks at the same location for a nucleotide different from the expected one.
What is the significance of having two peaks on the DNA sequencing diagram for an individual?
It indicates homozygosity with polymorphism.
How does DNA sequencing reveal if an individual is homozygous for a mutated gene?
By displaying two peaks at a nucleotide location where there should be one.
What does it suggest when a DNA sequencing diagram displays two peaks at the same position for an individual?
The individual has two different alleles for the same gene.
How is a homozygous mutation distinguished from a heterozygous polymorphism in DNA sequencing results?
By identifying overlapping bands at specific locations.
What does it indicate when two bands appear at the same level in columns A and T?
The person has a mutation in both chromosomes
What does it mean if a person is homozygous for a specific nucleotide sequence at a certain level?
The person has a mutation in both chromosomes
In DNA sequencing, what does it indicate when two individuals have the same nucleotide at a specific position on both chromosomes?
Both individuals have a mutation in one chromosome
If an individual shows a band at different levels in columns A and T, what does this suggest?
The individual has a polymorphism in one chromosome
What does it mean when a person has two bands at different levels in columns A and T?
The person is heterozygous for the mutation
In DNA sequencing, what does it suggest if an individual has different nucleotides on their maternal and paternal chromosomes at the same position?
The individual has a polymorphism in one chromosome
What is the main reason that incorporation of a dideoxynucleotide stops further DNA synthesis?
It lacks a 3' hydroxyl group for the addition of the next nucleotide
What is the role of a primer in the Sanger sequencing method?
To initiate DNA synthesis at a known sequence
In Sanger sequencing, why does the addition of a dideoxynucleotide terminate DNA synthesis?
It prevents elongation due to lack of a 3' hydroxyl group
How are different lengths of DNA fragments generated in Sanger sequencing?
By stopping synthesis at different points using dideoxynucleotides
What determines the size of each DNA fragment in Sanger sequencing?
The terminal dideoxynucleotide added to the growing chain
Why is gel electrophoresis used after Sanger sequencing reactions?
To separate DNA fragments based on size
What is the significance of labeling the primer with radioactive phosphorus in DNA sequencing?
To visualize the attached fragment on X-ray film
How does the Sanger sequencing method determine the order of nucleotides in a DNA sequence?
By reading the terminal dideoxynucleotides of separated fragments
How does gel electrophoresis aid in interpreting DNA sequences?
By separating fragments based on size for visualization
What does each labeled DNA molecule represent in Sanger sequencing?
A specific nucleotide sequence
Learn about the process of determining nucleotide order in a genome or DNA fragment, the importance of identifying genes, protein structure, and function. Understand how to read the DNA sequence from 5’ to 3’ direction.
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