Chapter 4: DNA Manipulation

Question 1

What is the general function of endonucleases in DNA manipulation?

Answer 1

Endonucleases cut DNA at specific recognition sites, and they are essential for manipulating DNA by cleaving the phosphodiester bond of the sugar-phosphate backbone.

Question 2

What is the significance of restriction endonucleases in DNA manipulation?

Answer 2

Restriction endonucleases, often sourced from bacteria, act as “molecular scissors” by cutting DNA at specific recognition sites. They are crucial for techniques like restriction endonuclease digestion.

Question 3

How are recognition sites of restriction endonucleases typically structured?

Answer 3

Recognition sites are usually palindromes, meaning the 5’ to 3’ sequence of the template strand is the same as the 5’ to 3’ sequence of the non-template strand.

Question 4

Differentiate between sticky ends and blunt ends created by endonucleases.

Answer 4

Sticky ends result from a staggered cut, while blunt ends come from a straight cut. Sticky ends have overhanging nucleotides, making them advantageous for inserting genes in DNA manipulation.

Question 5

What is the role of ligases in DNA manipulation?

Answer 5

Ligases join two fragments of DNA or RNA together by catalyzing the formation of phosphodiester bonds. They act like “molecular glue.”

Question 6

How do ligases differ from restriction endonucleases in terms of specificity?

Answer 6

Ligases lack specificity compared to restriction endonucleases. They can join any blunt or sticky ends, as their substrates are the sugar and phosphate groups of DNA or RNA.

Question 7

What is the function of polymerases in gene manipulation?

Answer 7

Polymerases synthesize polymer chains from monomer building blocks. DNA polymerase is particularly used in the replication or amplification of DNA.

Question 8

Why is a primer required for polymerase activity?

Answer 8

A primer is necessary for polymerase attachment to the template strand. Primers are short, single-stranded chains of nucleotides that are complementary to the template strand.

Question 9

Summarize the actions of restriction endonucleases, ligases, and polymerases in DNA manipulation.

Answer 9

Restriction Endonucleases: Cut DNA at specific sites.
Ligases: Join fragments of DNA or RNA.
Polymerases: Amplify sections of DNA or RNA.

Question 10

What is CRISPR-Cas9?

Answer 10

A defense system in bacteria against viral attacks. In gene editing, it’s a technology using Cas9 enzyme guided by synthetic sgRNA to modify specific DNA sequences.

Question 11

Explain the steps in the CRISPR-Cas9 defense system when a bacterium faces a virus.

Answer 11

Exposure: Virus injects DNA into bacterium.
Expression: CRISPR spacers transcribed into gRNA.
Extermination: Cas9-gRNA complex scans and cleaves complementary viral DNA.

Question 12

What is the role of Protospacer Adjacent Motif (PAM) in CRISPR-Cas9?

Answer 12

PAM is a specific DNA sequence near the target recognized by Cas9. It signals extraction of protospacer and enhances Cas9’s efficiency.

Question 13

How does CRISPR-Cas9 function in gene editing?

Answer 13

sgRNA Creation: Lab synthesizes sgRNA complementary to target DNA.
Cas9 Addition: Cas9 enzyme obtained with an appropriate PAM sequence.
Complex Formation: sgRNA and Cas9 mixed to form CRISPR-Cas9.
Cell Injection: Mixture injected into a cell, e.g., zygote.
DNA Cutting: Cas9 cuts target DNA, inducing repair and potential gene modification.

Question 14

What are applications and limitations of CRISPR-Cas9?

Answer 14

Applications: Research, disease treatment, and agriculture.
Limitations: Off-target effects, challenges in precise nucleotide substitutions, and ethical concerns about embryo modification and access equality.

Question 15

What is the purpose of the polymerase chain reaction (PCR)?

Answer 15

To amplify specific genes in a DNA sample, making it more efficient for further analysis.

Question 16

Describe the process of the polymerase chain reaction (PCR).

Answer 16

Denaturation: DNA heated to 90–95 °C, breaking hydrogen bonds and forming single-stranded DNA.
Annealing: DNA cooled to 50–55 °C, allowing primers to bind to complementary sequences.
Elongation: DNA heated to 72 °C, enabling Taq polymerase to synthesize a complementary DNA strand.
Repeat: Cycle (steps 1–3) is repeated to create multiple copies of DNA.

Question 17

What materials are required for the polymerase chain reaction (PCR)?

Answer 17

DNA Sample: Denatured and amplified during PCR.
Taq Polymerase: Enzyme binding complementary nucleotides to single-stranded DNA.
Nucleotide Bases: Constantly available for Taq polymerase to create a new DNA strand.
Sequence-Specific DNA Primers: Join to the 3’ end of single-stranded DNA, facilitating the formation of double-stranded DNA.

Question 18

Explain the importance of forward and reverse primers in PCR.

Answer 18

Forward primer binds to the start codon of the template strand, facilitating DNA synthesis in the same direction as RNA polymerase. Reverse primer binds to the stop codon of the coding strand, allowing DNA synthesis in the reverse direction. Both primers are essential for directional synthesis by Taq polymerase.

Question 19

How does the number of double-stranded DNA molecules change with each PCR cycle?

Answer 19

The number of double-stranded DNA molecules (x) per cycle can be determined using the formula x = 2^n, where n is the number of cycles. With each cycle, the amount of DNA doubles.

Question 20

What is the primary purpose of the polymerase chain reaction (PCR)?

Answer 20

The primary purpose of PCR is to amplify a DNA sample by creating multiple identical copies for further analysis.

Question 21

Why is the polymerase chain reaction (PCR) employed by scientists?

Answer 21

Scientists use PCR when there is an insufficient amount of a DNA sample for testing, enabling the amplification of DNA for various analyses.

Question 22

List some analyses that can be performed on DNA samples after undergoing the polymerase chain reaction (PCR).

Answer 22

Paternity Testing
Forensic Testing of Bodily Fluids
Analysis of Gene Fragments for Genetic Diseases

Question 23

What is the purpose of gel electrophoresis in the context of DNA manipulation?

Answer 23

Gel electrophoresis separates DNA fragments based on their molecular size, allowing scientists to analyze the composition and size of DNA fragments in a sample.

Question 24

Explain the steps involved in the process of gel electrophoresis.

Answer 24

Loading: DNA samples are placed in wells at one end of an agarose gel.
Electrophoresis: An electric current is passed through the gel, causing DNA fragments to move towards the positive electrode.
Separation: Smaller DNA fragments move faster through the gel, resulting in separation based on size.
Visualization: The gel is stained with a fluorescent dye, and DNA bands are visualized under UV light.

Question 25

Why is a standard ladder of DNA fragments with known sizes loaded alongside the samples in gel electrophoresis?

Answer 25

A standard ladder helps estimate the size of unknown DNA fragments by comparing their migration distance with fragments of known sizes in the ladder.

Question 26

What role does the electric current play in gel electrophoresis?

Answer 26

The electric current causes DNA fragments to move through the gel towards the positive electrode based on their size, facilitating their separation.

Question 27

Explain why smaller DNA fragments travel further in gel electrophoresis compared to larger fragments.

Answer 27

Smaller DNA fragments can move more easily through the pores in the agarose gel, covering more distance during electrophoresis, while larger fragments encounter more resistance.

Question 28

How is DNA visualized in gel electrophoresis, and why is it necessary?

Answer 28

DNA is visualized using a fluorescent dye like ethidium bromide, which binds to DNA fragments. Visualization is necessary because DNA is not visible to the naked eye, and the dye allows the bands to be seen under ultraviolet (UV) light.

Question 29

What are short tandem repeats (STRs), and how are they used in DNA profiling?

Answer 29

STRs are short, repeated sequences of nucleotides found in non-coding regions of DNA. They vary in length between individuals and are used in DNA profiling by analyzing their variations to create a unique DNA fingerprint for identification purposes.

Question 30

Explain the significance of a standard ladder in gel electrophoresis.

Answer 30

A standard ladder contains DNA fragments of known sizes, allowing scientists to estimate the size of unknown DNA fragments by comparing their migration distances to the ladder’s fragments.

Question 31

Describe the process of gel electrophoresis, outlining its key steps.

Answer 31

Loading Samples: DNA samples are placed in wells at one end of the gel.
Applying Electric Current: An electric current is applied, causing DNA fragments to move through the gel.
Separation Based on Size: Smaller fragments move faster and travel further, leading to separation based on size.
Visualization: The gel is stained, typically with ethidium bromide, and bands of DNA are visualized under UV light.

Question 32

In genetic testing for disorders like cystic fibrosis, why is it important to use specific DNA primers in the polymerase chain reaction?

Answer 32

Specific DNA primers are crucial in targeting and amplifying only the specific region of the gene associated with the disorder, ensuring accurate detection without amplifying the entire genome.

Question 33

How does gel electrophoresis contribute to genetic testing, particularly in diagnosing disorders like cystic fibrosis?

Answer 33

Gel electrophoresis helps visualize and analyze DNA fragments, enabling the identification of mutations or variations associated with genetic disorders, such as the presence of specific bands in the gel indicating the presence of mutated alleles.

Question 34

Explain the role of bacterial transformation in producing human insulin.

Answer 34

Bacterial transformation inserts recombinant plasmids into bacteria.
These plasmids carry insulin genes (subunits A and B).
Transformed bacteria express insulin proteins.

Question 35

Significance of plasmids in recombinant protein production.

Answer 35

Plasmids are circular DNA vectors.
They introduce foreign genes into bacteria.
Enable self-replication and carry antibiotic resistance genes.
Crucial for cost-effective protein synthesis.

Question 36

Process of creating recombinant plasmids for insulin.

Answer 36

Insulin genes inserted into plasmid vectors.
Restriction endonucleases cut both DNA types.
Sticky ends formed for gene integration.
DNA ligase joins genes and vectors.
Creates recombinant plasmids for insulin production.

Question 37

Why are bacteria commonly used in genetic modification?

Answer 37

Bacteria have plasmids for independent DNA replication.
Genetic modification exploits bacteria’s ability to take up foreign DNA.
Allows cost-effective production of various proteins.

Question 38

What are the key components of a plasmid vector?

Answer 38

Restriction Sites: Recognized by restriction endonucleases.
Antibiotic Resistance Genes: Identify transformed bacteria.
Origin of Replication (ORI): Signals DNA replication start.
Reporter Gene: Identifiable phenotype for successful vectors.

Question 39

Explain the role of a reporter gene in distinguishing recombinant and non-recombinant plasmids.

Answer 39

Reporter Gene: Encodes a trait (e.g., fluorescence) easily identified.
Recombinant Plasmid: Contains cut reporter gene due to gene of interest insertion.
Non-Recombinant Plasmid: Reporter gene is continuous.
Allows selection of transformed bacteria.

Question 40

Describe the process of bacterial transformation.

Answer 40

Uptake of Recombinant Plasmids: Facilitated by heat shock or electroporation.
Antibiotic Selection: Transformed bacteria identified on antibiotic-rich plates.
Reporter Gene Function: Distinguishes between recombinant and non-recombinant plasmids.

Question 41

How is insulin produced using recombinant plasmids?

Answer 41

Recombinant Plasmids: Contain insulin genes (subunits A and B).
Transformation: Bacteria take up plasmids, express insulin proteins.
Protein Extraction: Insulin proteins isolated and purified.
Combination: Alpha and beta subunits mixed to form functional human insulin.

Question 42

What is the quaternary structure of the insulin protein?

Answer 42

Two Subunits: Alpha and beta subunits.
Joined by Disulfide Bonds: Essential for insulin’s function.
Produced Separately: Using recombinant plasmids for each subunit.

Question 43

Explain the importance of the reporter gene (e.g., lacZ) in insulin production.

Answer 43

Protection: ß-galactosidase enzyme shields insulin subunit proteins.
Distinguishing: Reporter gene helps identify successful transformation.
Functional Testing: ß-galactosidase activity used to confirm gene expression.

Question 44

How is the insulin subunit isolated from the fusion protein?

Answer 44

Cyanogen Bromide: Added to break down the methionine added at the gene’s start.
Separation: Insulin subunit separated from ß-galactosidase.
Purification: Insulin subunit purified for combination with the other subunit.

Question 45

What is the significance of using synthetic DNA or cDNA in creating the gene of interest?

Answer 45

Intron Exclusion: Bacterial gene expression doesn’t involve introns.
Synthetic DNA: Lab-made genes lack introns.
cDNA: Reverse-transcribed from mRNA, naturally lacks introns.

Question 46

Compare heat shock and electroporation in bacterial transformation.

Answer 46

Heat Shock: Involves temperature changes to enhance membrane permeability.
Electroporation: Uses an electric shock for increased membrane permeability.
Purpose: Both methods facilitate recombinant plasmid uptake.

Question 47

Define genetic engineering and its goal in altering an organism’s genome.

Answer 47

Genetic Engineering: Process using biotechnology to alter an organism’s genome.
Goal: Confer desirable traits like increased size, drought resistance, or coloration.
Methods: Involves altering genes by silencing, insertion, removal, or nucleotide replacement.

Question 48

What is a genetically modified organism (GMO), and what is the role of the host organism in this process?

Answer 48

GMO Definition: Organism with altered genetic material using genetic engineering.
Host Organism: Receives altered gene/s, aims to confer desirable traits lacking in its genome.
Purpose: For research, commercial reasons, or specific advantageous characteristics.

Question 49

Differentiate between cisgenic and transgenic organisms.

Answer 49

Cisgenic Organisms: Genetically modified with genes from the same species (cisgenesis).
Transgenic Organisms: Genetically modified with genes from a different species (transgenesis).
Result: Both fall under the umbrella of genetically modified organisms (GMOs).

Question 50

How are transgenic organisms beneficial in agriculture, especially for crop improvement?

Answer 50

Protein Production: Transgenic organisms can produce proteins not part of their proteome.
Application: Used in agriculture for increased crop productivity and disease resistance.
Example: Golden rice, with increased vitamin A, addressing malnutrition.

Question 51

Explain the process of creating transgenic plants in agriculture.

Answer 51

Gene Identification: Identify and isolate a gene of interest from another species.
Gene Delivery: Insert the isolated gene into the host plant’s cells using a plasmid or direct insertion.
Gene Expression: Cultivate transformed cells, allowing the host to express the new transgene for agricultural use.

Question 52

How do GMOs contribute to increased crop productivity, and why is this significant?

Answer 52

Increased Yield: GMO technology increases crop yield per unit of land.
Importance: Essential to meet growing global food demand, especially in developing countries.
Challenges: Conventional breeding may not keep up with population growth; GMOs fill the gap.

Question 53

What is the role of GMOs in enhancing disease resistance in crops?

Answer 53

Disease Resistance: GMOs designed to resist harmful plant pathogens.
Food Security: Minimizes crop destruction and spreading of diseases, ensuring food security.
Additional Benefits: Can enhance resistance to other environmental factors like drought and pests.

Question 54

What does integrity entail in scientific research?

Answer 54

Integrity involves a commitment to searching for knowledge, reporting results honestly (favorable or unfavorable), and contributing to public understanding, permitting scrutiny.

Question 55

Define justice in the context of bioethics.

Answer 55

Justice is the moral obligation to ensure fair consideration of competing claims, prevent unfair burdens on specific groups, and ensure fair distribution and access to the benefits of an action.

Question 56

What is the commitment of beneficence in ethical decision-making?

Answer 56

Beneficence involves maximizing benefits and minimizing risks and harms associated with a particular position or course of action.

Question 57

Explain the concept of non-maleficence in scientific research.

Answer 57

Non-maleficence requires avoiding disproportionate harm from any position or action, ensuring that harm does not outweigh the benefits.

Question 58

What does respect involve in bioethical considerations?

Answer 58

Respect includes recognizing the intrinsic and instrumental value of living things, considering welfare, autonomy, beliefs, and cultural heritage, and empowering and protecting individuals with diminished decision-making capacity.

Question 59

Describe the focus of a consequence-based approach in ethics.

Answer 59

A consequence-based approach emphasizes considering the outcomes of an action, aiming for the maximization of positive results and minimization of negative results, focusing on the ends justifying the means.

Question 60

What is the core concept of a duty-based approach in ethics?

Answer 60

A duty-based approach prioritizes ethical rules, emphasizing that certain rules must be followed irrespective of potential consequences, rejecting causing immediate harm for a potential ‘greater good.’

Question 61

Explain the focus of a virtue-based approach in ethical decision-making.

Answer 61

A virtue-based approach considers the moral character of the person, emphasizing the characteristics and behaviors a ‘good’ person would exhibit, focusing on personal virtues rather than just actions.