BIOTECHNOLOGY

"the role of restriction enzymes in recombinant DNA technology."

"Restriction enzymes cleave (cut) DNA molecules at specific sequences, which is essential for manipulating DNA in recombinant DNA technology."

"Explain the process of nucleic acid hybridization."

"Nucleic acid hybridization allows for the locating of a specific DNA or RNA molecule with a high degree of accuracy by using complementary sequences."

"How can DNA molecules be amplified for study?"

"DNA molecules can be cloned or amplified via PCR (Polymerase Chain Reaction) to create concentrated samples containing billions of copies of the same sequence."

"Define a Southern blot procedure."

"A Southern blot is a method used to transfer DNA from an agarose gel to a membrane for the purpose of detecting specific DNA sequences using labeled probes."

"What is the purpose of using a radioactive probe in the Southern blot procedure?"

"A radioactive probe is used to hybridize with the target DNA sequence, allowing for the detection of the presence of that specific gene."

"Explain the difference between genomic and cDNA libraries."

"Genomic libraries contain at least one copy of all sequences in the genome of interest, while cDNA libraries contain complementary DNA copies made from mRNAs, representing genes that were actively transcribed at the time of collection."

"How is a genomic library constructed?"

"A genomic library is constructed by extracting genomic DNA, cutting it with restriction enzymes, and ligating the fragments into vectors for cloning."

"What is the significance of using less specific restriction enzymes in genomic library construction?"

"Using less specific restriction enzymes allows for more cuts in the DNA, resulting in a greater number of fragments, which can be beneficial for creating a comprehensive genomic library."

"Describe the process of screening a DNA library."

"Screening a DNA library involves using a radioactive probe to identify specific DNA sequences within the cloned fragments."

"What are the main types of DNA libraries?"

"The two main types of DNA libraries are genomic libraries and complementary DNA (cDNA) libraries."

"Explain the purpose of constructing a DNA library after confirming the presence of a gene."

"Constructing a DNA library allows researchers to store and analyze the genetic material of interest, facilitating further studies on gene function and expression."

"What is the role of agarose gel in the Southern blot procedure?"

"Agarose gel is used to separate DNA fragments based on size after digestion with restriction enzymes, allowing for the visualization of the DNA before transfer to a membrane."

"How does one locate a gene of interest in different species?"

"To locate a gene of interest in different species, one must isolate the gene, digest DNA from various species, and use a probe to identify the presence of the gene."

"What is the function of a cloning vector in genomic library construction?"

"A cloning vector, typically a plasmid, is used to package DNA fragments for transformation into bacteria, allowing for the replication and storage of the cloned DNA."

"Describe the significance of monitoring gene expression in different cell types."

"Monitoring gene expression in different cell types helps researchers understand how genes are regulated and function in various biological contexts."

"What is the purpose of incubating the membrane with hybridization buffer and labeled probe in the Southern blot procedure?"

"Incubating the membrane with hybridization buffer and labeled probe allows the probe to bind to its complementary DNA sequence, facilitating detection."

"Describe the process of using partial digestion with a restriction enzyme."

"Partial digestion with a restriction enzyme produces DNA fragments of appropriate size for constructing a genomic library."

"Explain the significance of using DNA probes in genomic libraries."

"DNA probes are used to screen plasmid or cosmid genomic libraries for specific DNA sequences, allowing researchers to identify the presence of desired sequences."

"Define chromosome walking in the context of genetic research."

"Chromosome walking is a technique used to identify and isolate DNA sequences by sequentially probing adjacent regions of a chromosome."

"How does the number of colonies needed for screening depend on the size of the inserted fragment?"

"The number of colonies required depends on the size of the inserted fragment, the size of the genome, and the desired probability of finding the fragment."

"What is cDNA and how is it produced?"

"cDNA, or complementary DNA, is produced from mRNA through a process called reverse transcription."

"Explain the role of reverse transcriptase in the production of cDNA."

"Reverse transcriptase is a protein that synthesizes a DNA copy of an RNA genome, generating cDNA from mRNA."

"Describe the characteristics of cDNA compared to mRNA."

"cDNA does not contain introns, as it is synthesized from processed mRNA, and it reflects which genes are expressed in specific cells or tissues."

"What is the first step in the process of generating cDNA from mRNA?"

"The first step is to isolate mRNA from cells."

"How do retroviruses utilize reverse transcriptase?"

"Retroviruses use reverse transcriptase to create a DNA copy of their RNA genome."

"Explain the importance of library screening in genetic research."

"Library screening allows scientists to clone DNA and identify individual genes within a genomic library."

"Describe the process of creating cDNA from mRNA."

"Using reverse transcriptase and a poly T primer, cDNA is synthesized from the mRNA."

"Explain the advantage of a cDNA library over a genomic library."

"A cDNA library provides the entire protein coding sequence, while a genomic library only contains the part of the sequence that matches the probe."

"How do microarrays function in gene expression analysis?"

"Microarrays allow for the detection of which genes are expressed in specific cells at a given time and can estimate levels of gene expression."

"Outline the steps involved in preparing a microarray."

"1. Spot cDNA clones on a glass plate. 2. Isolate cDNA from cells or tissues. 3. Label cDNAs with a fluorescent probe. 4. Denature cDNA and smear it on the microarray plate. 5. Use a laser to read the plate."

"What do different colors in microarray results indicate?"

"Green indicates healthy cells, red indicates tumor cells, and yellow indicates genes expressed in both cell types."

"How can densitometry be used in gene expression studies?"

"Densitometry estimates relative levels of gene expression, helping to identify differences between healthy and tumor cells."

"Define genomics and its significance."

"Genomics is the study of genomes, allowing for the sequencing of entire genomes and understanding genetic information."

"What is structural genomics?"

"Structural genomics involves sequencing genomes and analyzing nucleotide sequences to identify genes and regulatory elements."

"Summarize the timeline of the Human Genome Project."

"In 1986, NIH sought funding; work began in 1990; Celera Genomics was incorporated in 1998; Chromosome 1 was published in 1999; a working draft was announced in 2000."

"Describe the whole-genome sequencing (WGS) method."

"WGS involves cutting genomic DNA into fragments, sequencing them, and using computer programs to assemble the entire chromosome from aligned fragments."

"Describe the direct shotgun approach in genomic sequencing."

"The direct shotgun approach involves obtaining the genomic DNA sequence of an organism by randomly fragmenting the DNA and then sequencing these fragments to reconstruct the entire genome."

"Define contigs in the context of genomic DNA."

"Contigs are continuous fragments formed by overlapping segments of DNA that collectively create one continuous DNA molecule within a chromosome."

"Explain the role of algorithm-based software in genomic sequencing."

"Algorithm-based software programs are used to create DNA-sequence alignments, identifying overlapping sequences to help scientists reconstruct their order in the chromosome."

"How does high-throughput sequencing contribute to genomics?"

"High-throughput sequencing, facilitated by computer-automated DNA sequencers, allows for the rapid sequencing of large amounts of DNA, making projects like the Human Genome Project feasible."

"Describe the clone-by-clone approach to DNA sequencing."

"The clone-by-clone approach involves mapping DNA fragments from restriction digests to create a restriction map, ligating these fragments into vectors, and sequencing contigs to assemble the entire chromosome."

"What factors influence the decision to designate a genome sequence as 'final'?"

"The decision to designate a genome sequence as 'final' is influenced by the acceptable level of error that genome scientists are willing to accept."

"Explain the process of pyrosequencing."

"Pyrosequencing involves adding a DNA template, a primer, and one dNTP at a time, where the incorporation of the dNTP releases light, indicating successful sequencing."

"What is the significance of the brightness of the light in pyrosequencing?"

"The brightness of the light flash in pyrosequencing is proportional to the number of bases incorporated by the DNA polymerase, providing a measure of the sequencing progress."

"List the enzymes involved in pyrosequencing and their functions."

"The enzymes involved in pyrosequencing include: E1 - DNA polymerase (adds bases), E2 - ATP sulfurylase (converts PPi to ATP), E3 - Luciferase (converts ATP to oxyluciferin, releasing light), and E4 - Apyrase (degrades unincorporated nucleotides)."

"How does the clone-by-clone approach differ from the shotgun approach in genomic sequencing?"

"The clone-by-clone approach is a map-based method that involves creating a restriction map and aligning fragments, while the shotgun approach randomly fragments DNA and sequences it without prior mapping."

"Describe the structure and function of nanopores in nanopore sequencing."

"Nanopores are proteins embedded in a membrane with a hollow center that allows DNA molecules to pass through, enabling the sequencing of DNA."

"Explain the process of preparing plates for nanopore sequencing."

"Plates can be prepared with tens of thousands of individual nanopores, allowing for the simultaneous sequencing of multiple DNA fragments."

"How does the current assist in nanopore sequencing?"

"A current is used to 'drag' DNA fragments through the nanopores, facilitating their passage for sequencing."

"Define the role of enzymes in nanopore sequencing."

"An enzyme creates a hairpin loop on one end of a DNA fragment and separates the strands on the other end as the fragment is drawn through the nanopore."

"What unique signals do nucleotides produce during nanopore sequencing?"

"Each of the four nucleotides (G, A, T, and C) produces a unique electrical signal as they pass through the nanopore, which is used to determine the DNA sequence."

"Explain the data output of nanopore sequencing."

"Nanopore sequencing produces a vast amount of data in a very short time, which is then analyzed using computer programs."

"Discuss the specificity of nanopore sequencing compared to traditional methods."

"Nanopore sequencing lacks specificity as genomic DNA is inputted and all available sequences are read, unlike PCR products or cloned DNA fragments."

"Outline the current method of gene sequencing using PCR."

"1. Extract DNA 2. Use PCR to amplify the gene of interest 3. Check results with gel electrophoresis 4. Perform sequencing reaction 5. Run on sequencer, resulting in the sequence of the gene of interest in about 24 hours."

"How can nanopore sequencing be used to obtain an entire genome?"

"By repeating the sequencing process multiple times, the entire genome can be obtained in just a few hours, with computer analysis used to locate the gene of interest."

"Describe the first step in positional cloning."

"The first step is to perform RFLPs (Restriction Fragment Length Polymorphisms) to identify a restriction fragment linked to a disease allele."

"What is the significance of SNPs in the context of positional cloning?"

"With the Human Genome sequenced, SNPs (Single Nucleotide Polymorphisms) can now be used in positional cloning to identify genetic differences."

"Explain the purpose of in situ hybridization in positional cloning."

"In situ hybridization is used to determine which chromosome a specific RFLP fragment is located on."

"What is the goal of locating known RFLP sites during positional cloning?"

"The goal is to find known RFLP sites on either side of the locus of interest to assist in mapping and identifying the disease allele."

"Describe the process of linkage analysis in positional cloning."

"Linkage analysis involves mapping RFLP sites and known restriction sites to identify the region of interest related to a genetic disease."

"What is chromosome walking in the context of genetic mapping?"

"Chromosome walking is a technique used to identify and map genes by sequentially locating RFLP sites along a chromosome."

"Describe the process of gene therapy for treating diseases caused by mutant proteins."

"Gene therapy involves inserting a functional (non-mutant) copy of a gene into the genome of an afflicted organism to replace the defective gene causing the disease."

"Explain the two types of gene therapy."

"The two types of gene therapy are somatic cell therapy, which transforms somatic cells with a functional copy of a gene, and germ line therapy, which transforms germ cells (sperm and egg) with a functional copy of a gene."

"How are somatic cell diseases treated in gene therapy?"

"Somatic cell diseases are treated by transforming somatic cells with a functional copy of the gene, making them easier to address compared to germ line diseases."

"Identify the steps involved in treating SCID using gene therapy."

"1. Isolate T-Cells from the afflicted patient. 2. Use a retrovirus to transform T-Cells by adding a functional copy of the ADA gene. 3. Reintroduce the transformed T-cells into the patient."

"What is the challenge associated with gene therapy for autoimmune diseases?"

"The challenge is how to effectively deliver the functional copy of the gene into the appropriate cells."

"Describe the role of bone marrow transplants in gene therapy for Sickle Cell Anemia."

"Bone marrow transplants involve replacing the patient's bone marrow with stem cells from the umbilical cord of a newborn, which can help treat Sickle Cell Anemia."

"Explain the delivery challenges faced in gene therapy for systemic diseases."

"Systemic diseases are more difficult to treat because the challenge lies in delivering the gene to the specific cells that need it."

"How is gene therapy applied in the treatment of Cystic Fibrosis?"

"In Cystic Fibrosis, a functional copy of the gene coding for the enzyme needed to break down mucus is inserted into a virus, which the patient inhales as an aerosol, allowing the viral DNA to infect lung cells and produce the functional enzyme."

"What is the significance of the AV node in heart disease treatment?"

"The AV node regulates heart rhythm, and gene therapy can target it to address arrhythmias by delivering mRNA for the inhibitory G-protein gene."

"List the treatment options for heart attacks."

"The treatment options for heart attacks include angioplasty and bypass surgery."

"How can gene therapy stimulate new blood vessel growth in blocked arteries?"

"Gene therapy can stimulate new blood vessel growth by delivering genes that promote angiogenesis to the blocked blood vessels."

"What is the purpose of the vector in gene therapy?"

"The vector carries a non-functional gene of interest (GOI) and a neomycin resistance gene, flanked by homologous regions to facilitate gene knockout."

"Describe the steps involved in creating a culture of embryonic stem (ES) cells for gene therapy."

"Step 1: Create a culture of embryonic stem (ES) cells. Step 2: Add the vector to the culture dish."

"What are the possible outcomes when adding a vector to embryonic stem cells?"

"1. Cells fail to incorporate the vector. 2. Cells successfully incorporate the vector."

"Describe the process of incorporating a vector into host cells."

"Cells can incorporate a vector at a random location or into the correct location, effectively 'knocking out' the host cell's gene."

"Explain the two levels of selection used in creating knockout mice."

"The first level involves adding neomycin to block protein synthesis, killing cells without the vector. The second level involves adding ganciclovir, which, when combined with the tk gene product, produces a toxic compound that kills cells."

"How are ES cells introduced into developing embryos in the knockout mouse process?"

"ES cells are inserted into developing embryos and then implanted into pseudopregnant females."

"What is the purpose of crossing transgenic mice in the knockout process?"

"Crossing transgenic mice produces heterozygous males and females, which can then be crossed to yield homozygous knockout offspring."

"Define conditional knockouts in the context of genetic engineering."

"Conditional knockouts involve using the Cre-LoxP system, where Cre recombinase removes genes located between two LoxP sites, allowing for cell-specific gene knockout."

"Explain how Cre recombinase is used in cell-specific knockouts."

"Cre recombinase cuts DNA at specific loxP sites, allowing for the removal of genes in specific cell types when combined with an appropriate promoter."

"Describe the role of an inducible promoter in the Cre-LoxP system."

"An inducible promoter allows for the expression of Cre recombinase to be controlled, enabling the gene knockout to be activated by external factors like temperature changes or nutrient addition."

"How can a vector be constructed for a gene of interest (GOI) in cell-specific knockouts?"

"The vector should include the promoter region of the gene expressed in the target cell type, along with the coding region for the GOI, flanked by loxP sites."

"What happens when Cre recombinase is expressed in offspring from crossed mouse lines?"

"When Cre recombinase is expressed, it can disable the gene located between the loxP sites, allowing researchers to observe the effects of the knockout."

"Explain the significance of producing a second line of mice with Cre recombinase."

"Producing a second line with Cre recombinase allows for controlled gene knockout in specific tissues or at specific times, enhancing the precision of genetic studies."