Unit 3 AOS 1 Flashcards
What are endonucleases?
Endonucleases are enzymes that cut strands of DNA. They target specific recognition sites, and when they do, they break the phosphodiester bonds between nucleotides, resulting in a cleaved DNA strand.
What are restriction endonucleases and why are they important?
Restriction endonucleases are a subset of endonucleases that cut DNA at specific sequences known as recognition sites. They are typically derived from bacteria and act as a defense mechanism against viral DNA. For example, EcoRI, HindIII, and AluI are common restriction endonucleases.
What is a recognition site in DNA, and what is a palindrome in this context?
: A recognition site is a specific sequence of DNA to which a restriction endonuclease binds and cuts. A palindrome refers to the sequence being the same when read in both directions on opposite strands. For example, EcoRI cuts at the recognition sequence GAATTC, which is a palindrome.
What is the difference between sticky ends and blunt ends in DNA cutting?
Sticky ends result from staggered cuts in the DNA, leaving overhanging nucleotides, while blunt ends result from straight cuts with no overhanging nucleotides.
What is the effect of restriction endonucleases like EcoRI and AluI on DNA?
EcoRI creates sticky ends by cutting at a specific sequence (GAATTC), whereas AluI creates blunt ends by cutting at a different sequence (AGCT).
What do ligases do in DNA manipulation?
Ligases are enzymes that join two fragments of DNA (or RNA) by catalyzing the formation of phosphodiester bonds. They work in reverse to endonucleases, “gluing” DNA pieces together.
What is the difference between DNA ligase and RNA ligase?
DNA ligase joins DNA fragments, while RNA ligase joins RNA fragments. Both catalyze the formation of phosphodiester bonds but are specific to their substrate
How does DNA ligase work on sticky ends vs. blunt ends?
DNA ligase can join both sticky ends and blunt ends. For sticky ends, it joins complementary overhanging nucleotides, while for blunt ends, it directly connects the ends without overhangs.
What is the function of polymerases in DNA manipulation?
Polymerases are enzymes that synthesize polymer chains, such as creating new DNA strands from nucleotides. They play a crucial role in DNA replication and gene amplificat
What are the two main polymerases used in gene manipulation?
RNA polymerase, used for transcription, and DNA polymerase, used for replication or amplification of DNA.
How is DNA polymerase used in DNA amplification?
DNA polymerase synthesizes additional DNA strands to amplify small DNA samples, which is commonly used in techniques like PCR (Polymerase Chain Reaction) to create large quantities of DNA from small samples.
What role do primers play in DNA replication?
Primers are short strands of nucleotides that provide a starting point for DNA polymerase to attach and begin synthesizing the complementary strand in the 5’ to 3’ direction.
What are the three main types of enzymes used in DNA manipulation, and their functions?
Back:
Endonucleases: Cut DNA at specific recognition sites.
Ligases: Join DNA fragments by forming phosphodiester bonds.
Polymerases: Synthesize DNA or RNA by adding nucleotides to a template strand.
How can endonucleases, ligases, and polymerases be used to make a Mexican walking fish glow in the dark?
Endonucleases cut out the GFP gene from jellyfish DNA, polymerases amplify the GFP gene, and ligases insert it into the axolotl genome, making it glow in the dark.
What is the result of adding HindIII to the following DNA sequence:
5’ G G C C T A T G AAG C T T GAA 3’
HindIII cuts at the recognition site AAGCTT, creating two DNA fragments.
Which enzyme is used to join two DNA fragments together?
A. DNA polymerase
B. Ligase
C. Endonuclease
Ligase
What is the difference in how circular DNA and linear DNA are cut by restriction enzymes?
For circular DNA, the number of fragments created equals the number of recognition sites. For linear DNA, the number of fragments equals the number of recognition sites plus one.
What are ligases?
Ligases are enzymes that join two fragments of DNA or RNA together by catalyzing the formation of phosphodiester bonds, acting like “molecular glue.”
How do ligases function in genetic engineering?
Ligases catalyze the joining of DNA or RNA fragments, such as when DNA fragments are inserted into plasmids or when sticky and blunt ends are connected during cloning.
Q: How do ligases differ from endonucleases?
While endonucleases cut DNA, ligases join DNA or RNA fragments together. Ligases do not have the specificity of restriction endonucleases and can join both sticky and blunt ends.
How do DNA ligase and RNA ligase differ?
DNA ligase joins two DNA fragments, while RNA ligase joins RNA fragments. Both catalyze the formation of phosphodiester bonds to link the fragments.
What are the advantages of using sticky end ligation versus blunt end ligation?
Sticky end ligation has an advantage in ensuring that inserted genes are oriented correctly due to the complementary base pairing between the sticky ends, while blunt end ligation is more straightforward but less efficient.
What is the role of ligation in genetic modification?
Ligation is crucial for inserting foreign genes into plasmids or vectors by joining the gene of interest with the plasmid DNA, enabling the transfer of new genetic material into cells.
What is the action of DNA ligase on sticky and blunt end fragments?
DNA ligase joins sticky end fragments by matching overhanging nucleotides and also joins blunt end fragments by directly connecting the ends without overhangs.
What is the main function of RNA polymerase during gene expression
RNA polymerase transcribes DNA into RNA, which is then used by ribosomes to synthesize proteins, amplifying the gene.
What is the key difference between RNA polymerase and DNA polymerase
RNA polymerase synthesizes RNA from DNA, while DNA polymerase synthesizes new DNA strands during replication or amplification.
What is crispr
CRISPR is a naturally occurring sequence of DNA found in bacteria that plays an important role in their defence against viral attacks.
what is a bacteriophage
- a virus that infects prokaryotic organisms
How do bacteria defend themselves against viruses using CRISPR-Cas9?
Bacteria store viral DNA in their genome as “mugshots.” When a virus attacks again, bacteria transcribe this stored DNA and attach it to an enzyme called Cas9. This helps Cas9 destroy the invading viral DNA without harming the bacterium’s own DNA.
What is the role of Cas9 in the CRISPR-Cas9 system?
Cas9 is an endonuclease that cuts the viral DNA, guided by the transcribed “mugshot” (spacer DNA), ensuring that only viral DNA is targeted and destroyed.
What are the three steps in the CRISPR-Cas9 defense system?
Exposure - the bacteriophage injects its DNA into a bacterium, which identifies
the viral DNA as a foreign substance. Cas1 and Cas2 are both CRISPR-associated
enzymes like Cas9, but they serve a different purpose
Expression - the CRISPR spacers are transcribed along with half a palindrome
from the repeat either side of it, and converted into an RNA molecule known as
guide RNA (gRNA). gRNA binds to Cas9 to create a CRISPR-Cas9 complex which
is directed t
Extermination -
scans the cell for invading bacteriophage DNA that is complementary to the ‘mugshot’ on the gRNA. After the CAS9 cleaves the phosphate sugar backbone to inactivate the virus
What is a spacer in the context of CRISPR-Cas9?
A spacer is a short segment of DNA that comes from a virus, inserted into the CRISPR sequence to serve as a “mugshot” of the virus. It helps the bacterium recognize and destroy the virus during future infections.
What is the function of the Cas1 and Cas2 proteins in the CRISPR-Cas9 system?
Cas1 and Cas2 are CRISPR-associated enzymes that cut the viral DNA into short segments (protospacers), which are then added to the CRISPR sequence as spacers for future recognition.
How does the CRISPR sequence appear in the bacterial genome
The CRISPR sequence consists of short, repeated nucleotide sequences (palindromic) interspersed with spacer DNA, which is derived from previous viral infections.
What is a protospacer
A protospacer is a short segment of DNA from an invading virus that is cut by Cas1 and Cas2 and then inserted into the CRISPR sequence as a spacer for future virus recognition.
What are the components of the CRISPR-Cas9 system?
The CRISPR-Cas9 system consists of the CRISPR sequence (repeated and spacer DNA), the Cas9 endonuclease, and guide RNA (gRNA), which directs Cas9 to specific viral DNA sequences.
What is the significance of CRISPR-Cas9 in genetic engineering?
CRISPR-Cas9 has been modified by scientists to precisely edit the genomes of various organisms, making it a powerful tool for gene editing in research, medicine, and biotechnology.
What are palindromic sequences in the context of CRISPR
Palindromic sequences are short, repeated DNA sequences that are the same in both directions. These repeats are part of the CRISPR structure, helping to form the pattern needed for viral DNA recognition.
How does the CRISPR-Cas9 system help bacteria survive viral infections?
The system stores viral DNA in the form of spacers in the CRISPR sequence. When the same virus attacks again, the stored DNA helps the bacterium recognize and cut the viral DNA, preventing further infection.
Why is the CRISPR-Cas9 system considered a form of “adaptive immunity” in bacteria?
It is considered adaptive immunity because bacteria “remember” past viral infections by storing viral DNA as spacers in the CRISPR sequence. This allows the bacteria to recognize and defend against the same virus in the future.
Q: What is the primary advantage of using CRISPR-Cas9 for gene editing?
CRISPR-Cas9 allows for precise and targeted changes to the genome, making it a powerful and cost-effective tool for genetic engineering, unlike other methods that may lack precision.
What is sgRNA in gene editing?
sgRNA (single guide RNA) is a synthetic RNA created to guide Cas9 to a specific DNA sequence for editing. It is similar in function to the gRNA used in bacteria but differs in its structure.
What are some agricultural applications of CRISPR-Cas9?
: CRISPR-Cas9 can be used to:
Introduce pest and herbicide resistance to crops.
Alter genes to improve crop growth rates and yield.
What are some limitations of CRISPR-Cas9?
Limitations include:
Difficulty in ensuring precise insertion of new genetic material.
Ethical concerns about editing human embryos.
Safety risks such as off-target cleavages (unintended edits).
What ethical concerns are associated with CRISPR-Cas9?
Ethical concerns include:
The potential for unintended consequences or harm when editing embryos.
The issue of informed consent, as embryos cannot consent to genetic changes.
Inequality, as only wealthy individuals may have access to gene editing technologies.
Discrimination and societal pressure to “improve” genetics.
What is a gene knockout in the context of CRISPR-Cas9
A gene knockout involves disabling or silencing a gene to study its function or to understand the effects of not having that gene active.
What is the difference between CRISPR-Cas9 in bacteria and in gene editing?
In bacteria, CRISPR-Cas9 is used to defend against viral DNA by storing and using “mugshots” of the virus. In gene editing, CRISPR-Cas9 is used to make precise modifications in the DNA of organisms.
What is the PAM sequence in the CRISPR-Cas9 process?
The PAM sequence (Protospacer Adjacent Motif) is a short DNA sequence required for Cas9 to bind to the target DNA and initiate the cutting process.
What happens after CRISPR-Cas9 cuts the target DNA?
After the DNA is cut, the cell’s repair mechanisms are triggered, which can result in the insertion, deletion, or alteration of DNA at the cut site, leading to changes in gene function.
Why is CRISPR-Cas9 considered more efficient than other gene-editing tools?
CRISPR-Cas9 is more precise, affordable, and easier to use compared to other genetic engineering techniques, making it a popular choice for gene editing.
what is a zygote
te the diploid cell formed by
the combination of two haploid
gamete cells
What is the purpose of the Polymerase Chain Reaction (PCR)?
PCR is a technique used to amplify a small DNA sample by making multiple identical copies, enabling scientists to analyze DNA when there is not enough for testing.
Name three key applications of PCR.
Paternity testing
Forensic testing (e.g., analyzing DNA from crime scenes)
Analyzing gene fragments for genetic diseases
What materials are required for PCR to occur?
DNA sample
Taq polymerase enzyme
Nucleotide bases
Forward and reverse primers
What is the role of Taq polymerase in PCR?
Taq polymerase is a heat-resistant enzyme that synthesizes new strands of DNA by attaching complementary nucleotides to a single-stranded DNA template.
What is the role of primers in PCR?
: Primers are short DNA sequences that bind to complementary regions of the target DNA and provide a starting point for DNA synthesis by Taq polymerase.
What are the four steps of the PCR process
Denaturation:The DNA is heated to 90-95°C to break the hydrogen bonds between the complementary strands, resulting in two single-stranded DNA molecules.
Annealing: The mixture is cooled (50-55°C) so primers can bind to the DNA.
Elongation: DNA is heated to 72°C for Taq polymerase to synthesize new DNA strands.
Repeat: The cycle is repeated multiple times to amplify the DNA.
How many copies of DNA are made after 10 cycles of PCR?
1024 copies of the DNA are made after 10 cycles.
What is the difference between the forward and reverse primers in PCR?
The forward primer binds to the 3’ end of the template strand and guides Taq polymerase to synthesize a new DNA strand in the same direction as RNA polymerase.
The reverse primer binds to the 3’ end of the coding strand and guides Taq polymerase to synthesize a new DNA strand in the opposite direction.
Why is Taq polymerase preferred over human DNA polymerase in PCR?
Taq polymerase is heat-resistant and functions optimally at 72°C, which is necessary for the high temperatures used in PCR, while human DNA polymerase is not as heat-resistan
How does the number of DNA copies increase with each PCR cycle?
the amount of DNA doubles with each cycle. After the first cycle, there are 2 copies; after the second cycle, 4 copies; and so on, growing exponentially.
What is the significance of the PCR thermal cycler?
The thermal cycler automatically adjusts the temperature at each stage of the PCR process, ensuring precise control over the denaturation, annealing, and elongation steps.
What is the purpose of gel electrophoresis
Gel electrophoresis is used to separate DNA fragments based on their size. It helps measure the size of DNA fragments and is used in applications like genetic testing and DNA profiling.
What is a “standard ladder” in gel electrophoresis?
A standard ladder is a mixture of DNA fragments of known sizes. It is used as a reference to estimate the size of unknown DNA fragments by comparing their migration distance.
What is the role of agarose gel in gel electrophoresis?
Agarose gel is a sponge-like material with tiny pores. It allows DNA fragments to move through when an electric current is applied, separating them based on size.
Why is a buffer solution used in gel electrophoresis?
The buffer solution conducts the electric current through the agarose gel and helps maintain the pH of the environment, ensuring proper DNA migration.
What happens to DNA when an electric current is applied during gel electrophoresis?
DNA, which is negatively charged due to its phosphate backbone, moves towards the positive electrode when an electric current is applied.
How do the sizes of DNA fragments affect their movement through the gel?
Smaller DNA fragments move faster and travel further through the gel, while larger DNA fragments move slower and do not travel as far.
Why is ethidium bromide used in gel electrophoresis?
Ethidium bromide is a fluorescent dye that binds to DNA. It allows the DNA bands to be visualized under ultraviolet (UV) light since DNA is not visible to the naked eye.
What is the significance of the bands observed in the gel after electrophoresis?
: Each band represents a collection of DNA fragments of the same size. The bands are used to determine the number and size of DNA fragments in a sample.
How are gel electrophoresis results interpreted?
The size of the DNA fragments is estimated by comparing the distance they traveled to the known sizes in the standard ladder. Thicker bands indicate more DNA of that size.
What are Short Tandem Repeats (STRs), and how are they used in DNA profiling?
STRs are short, repetitive DNA sequences that vary in length between individuals. They are used in DNA profiling because they provide a unique genetic fingerprint that can help identify individuals or determine familial relationships.
What can gel electrophoresis reveal in genetic testing for disorders like cystic fibrosis?
Gel electrophoresis can detect mutations in genes, such as the CFTR gene in cystic fibrosis, by comparing the sample’s DNA fragment sizes to those of known healthy or mutated alleles.
: How can gel electrophoresis be applied in criminal investigations?
in forensic science, gel electrophoresis can be used to compare DNA samples from crime scenes to suspects’ DNA. It helps identify the perpetrator or establish familial relationships.
What does it mean if two samples have matching Short Tandem Repeats (STRs) in a criminal case?
: If two DNA samples have matching STRs, it suggests that the samples come from the same individual, which can help identify a suspect in a criminal investigation.
What factors can affect the movement of DNA fragments in gel electrophoresis?
Factors include the voltage applied, gel composition (density and agarose concentration), buffer concentration, and the time the electric current is applied.
What is bacterial transformation?
Bacterial transformation is the process by which bacteria take up foreign DNA (such as recombinant plasmids) from their environment.
What are two common methods of bacterial transformation?
Heat shock and electroporation are two common methods used to increase the permeability of the bacterial membrane, allowing recombinant plasmids to enter.
How do scientists distinguish between recombinant and non-recombinant plasmids in bacteria?
Reporter genes, like GFP (Green Fluorescent Protein), are used to distinguish recombinant plasmids. Non-recombinant plasmids will express the reporter gene, while recombinant plasmids will not.
What is a fusion protein, and why is it used in insulin production?
A fusion protein is a protein made by joining two separate proteins together. In insulin production, insulin subunits are fused with ß-galactosidase to protect them from digestive enzymes in bacteria.
What are some proteins produced through bacterial genetic modification?
Some examples include insulin, erythropoietin, chymosin, interferon, growth hormone, and alpha-amylase.
Why must the gene of interest be free of introns before being inserted into a plasmid?
Bacteria cannot process introns, so the gene of interest must be in a form without introns to be correctly expressed in bacteria.
How does the antibiotic selection process help identify transformed bacteria?
Only transformed bacteria containing recombinant plasmids will survive on plates with antibiotics, as the plasmids contain antibiotic resistance genes.
What is the role of the reporter gene in recombinant plasmid selection?
A reporter gene, like GFP, helps identify whether a plasmid contains the gene of interest by exhibiting a visible trait, such as fluorescence under UV light.
How is insulin produced in genetically modified bacteria?
: Insulin is produced by transforming bacteria with two recombinant plasmids—one containing the alpha subunit gene and the other containing the beta subunit gene. The subunits are then combined to form functional human insulin.
What is the purpose of adding methionine at the beginning of the insulin gene?
The methionine is added to facilitate the production of a fusion protein, protecting the insulin subunit from bacterial digestive enzymes.
What is electroporation?
Electroporation is a method where an electric shock is applied to bacterial cells to make their membrane more permeable, allowing recombinant plasmids to enter.
What is the difference between genetically modified organisms (GMOs) and transgenic organisms (TGOs)?
Genetically modified organisms (GMOs) are any organisms whose genetic material has been altered through genetic engineering techniques. Transgenic organisms (TGOs) are a specific type of GMO that contains genes from a different species, created through transgenesis, where foreign DNA is inserted into the organism’s genome.
hat are the two main types of GMOs
Cisgenic organisms, which have genes from the same species inserted into their genome.
Transgenic organisms, which have genes from a different species inserted into their genome, resulting in a genetically modified organism with foreign DNA.
What are some common agricultural uses of GMOs?
GMOs are commonly used in agriculture to:
Increase crop productivity (higher yields per unit of land),
Improve resistance to diseases and pests, reducing the need for chemical pesticides, and
Enhance nutritional value of crops (e.g., increasing vitamins or other nutrients in staple foods like rice)
How are transgenic plants created?
The process of creating transgenic plants typically involves three key steps:
Gene identification: A beneficial gene is identified and isolated from another organism.
Gene delivery: The gene is inserted into the plant’s genome using methods like gene guns or bacterial plasmids.
Gene expression: The modified plant grows and produces the desired protein or trait, which is expressed in the plant’s tissues.
What is Golden Rice, and why was it developed
Golden Rice is a genetically modified crop developed to combat vitamin A deficiency (VAD), which causes preventable blindness, particularly in children in developing countries. It was engineered to produce beta-carotene (a precursor of vitamin A) in the rice grains by inserting two genes: one from a daffodil and the other from a bacterium.
How do Bt crops help with pest resistance?
Bt crops are genetically modified to produce proteins from the bacterium Bacillus thuringiensis (Bt) that are toxic to specific pests. These toxins are safe for humans but kill insects that ingest them, providing built-in pest resistance and reducing the need for chemical pesticides.
Why is increasing crop productivity important?
Increasing crop productivity is essential to meet the growing global food demand due to population growth, which is expected to reach 9.2 billion by 2040. As the available arable land decreases and environmental challenges increase, GMOs can help increase crop yields, ensuring that sufficient food is available for everyone.
How does selective breeding differ from genetic engineering?
Selective breeding is the traditional method of choosing plants or animals with desirable traits to reproduce, passing these traits to future generations through natural processes. In contrast, genetic engineering directly manipulates an organism’s DNA to introduce new traits, often involving genes from different species.
What ethical concerns surround the use of GMOs?
Ethical concerns about GMOs include potential environmental risks such as crossbreeding with wild populations, unanticipated effects on ecosystems, and the concentration of food production in the hands of a few large corporations. There are also health concerns about the long-term effects of consuming GMOs and the impact on biodiversity.
How can GMOs help with food security in developing countries?
GMOs can help improve food security in developing countries by increasing crop yields, enhancing resistance to pests and diseases, and improving the nutritional value of staple crops. For example, Golden Rice provides a source of vitamin A to populations where deficiencies are common.
Biological and ethical implications of bt cotton and golden rice
Bt cotton Farmers benefit from reduced pesticide use, which lowers costs and improves safety. Over time, pests may develop resistance to the Bt toxin, reducing its effectiveness.
Golden Rice Golden Rice can help fight vitamin A deficiency in unwealthy regions, improving nutrition. The introduction of Golden Rice may affect local ecosystems by potentially cross-breeding with wild rice.
What role would DNA ligase play?
DNA ligase would join the DNA ends together after the Cas9 protein has cut the DNA. Allowing the new genetic material to be inserted or the edited DNA to be reconnected properly.
Explain why CRISPR-Cas9 would be beneficial for genetically engineering agricultural crops.
CRISPR-Cas9 allows for precise modifications to a crop’s DNA, improving desirable traits like disease resistance. This technology is faster and more accurate than traditional breeding, potentially increasing crop yi`
