genetics, dna cloning, and genome sequencing techniques

What is selective breeding?

A practice of choosing desirable traits in plants and animals to shape their forms, serving as an early form of genetic modification.

What do restriction enzymes do?

They cut DNA molecules at specific short sequences, often palindromic, producing either blunt or sticky ends.

What are sticky ends in DNA cloning?

Single-stranded overhangs produced by staggered cuts from restriction enzymes that can base-pair with complementary sequences.

How does gel electrophoresis work?

It separates DNA fragments by size, with smaller fragments migrating faster toward the positive electrode in an agarose gel.

What is a plasmid?

A small, circular, double-stranded DNA molecule used as a cloning vector in genetic engineering.

How is recombinant DNA created?

By cutting a plasmid and a DNA fragment with the same restriction enzyme, allowing them to base-pair and sealing them with DNA ligase.

What is the role of DNA ligase in cloning?

It seals the sugar-phosphate backbone of DNA fragments, forming a continuous circular DNA molecule in recombinant DNA.

How does DNA cloning in bacteria work?

A recombinant plasmid is introduced into a bacterial cell, which then replicates the plasmid as it divides, producing many copies.

What is a genomic library?

A collection of bacteria, each carrying a different fragment of human DNA, representing the entire human genome in smaller pieces.

How is complementary DNA (cDNA) synthesized?

From mRNA using reverse transcriptase and DNA polymerase, providing a DNA copy of expressed genes.

What is the significance of hybridization in molecular biology?

It allows complementary DNA or RNA strands to re-form a double helix after denaturation, essential for techniques like DNA hybridization.

What are the three main steps of the Polymerase Chain Reaction (PCR)?

Denaturation (heating to separate strands), annealing (binding primers), and extension (synthesizing new DNA strands).

What is the purpose of using heat-stable DNA polymerase in PCR?

To withstand the high temperatures required for DNA strand separation during the denaturation step.

What happens during the denaturation step of PCR?

Double-stranded DNA is heated to separate it into two single strands.

What is the role of primers in PCR?

Short DNA sequences that bind to complementary sequences flanking the target DNA region, initiating DNA synthesis.

What is the outcome of repeating the PCR cycle multiple times?

The amount of target DNA is doubled with each cycle, leading to exponential amplification.

What is the function of RNase in cDNA synthesis?

It partially degrades the RNA strand in the RNA-DNA hybrid, allowing DNA polymerase to synthesize the second DNA strand.

What is the significance of the 5′ ends in cDNA libraries?

They are often missing because cDNA is synthesized from the poly A tail of mRNA, which does not include the 5′ ends.

What is the main advantage of using plasmids as cloning vectors?

They can replicate independently inside bacteria, making them effective carriers for amplifying foreign DNA sequences.

What is the importance of comparing DNA fragments to size markers in gel electrophoresis?

It allows researchers to estimate the sizes of DNA fragments based on their migration distance in the gel.

What is the result of hybridization between complementary DNA strands?

The restoration of the double helix structure through the formation of hydrogen bonds.

What is the key feature of restriction enzymes that aids in genetic engineering?

Their ability to recognize and cut specific DNA sequences, allowing for precise manipulation of genetic material.

What is the purpose of PCR (Polymerase Chain Reaction)?

To amplify a specific DNA region through repeated cycles of heating, cooling, and DNA synthesis.

What role does thermostable DNA polymerase play in PCR?

It allows the reaction to be repeated many times without needing fresh enzyme, enabling the production of millions of copies of the target sequence.

What happens during the heating phase of PCR?

The DNA strands are separated.

What is the function of primers in PCR?

Primers anneal to the DNA strands during the cooling phase, providing a starting point for DNA synthesis.

How does the amount of DNA change with each cycle of PCR?

The amount of DNA doubles with each cycle, leading to exponential amplification.

What is the significance of the third cycle in PCR?

By the third cycle, some DNA fragments are the exact length of the target region defined by the primers.

What are genomic clones in PCR?

Clones generated by isolating total DNA from cells and selectively amplifying a specific DNA region of interest.

How are cDNA clones created in PCR?

By purifying total mRNA, synthesizing a complementary DNA (cDNA) strand from the mRNA template, and then amplifying it with PCR.

What diagnostic applications does PCR have?

PCR can detect viral genomes in blood samples, such as HIV, by amplifying viral RNA converted to cDNA.

What is the process of detecting HIV using PCR?

Blood is collected, plasma is isolated, RNA is extracted, converted to cDNA, amplified by PCR, and analyzed using gel electrophoresis.

What is the dideoxy sequencing method?

A DNA sequencing technique that uses chain-terminating nucleotides (ddNTPs) to halt DNA synthesis at specific points.

How does automated dideoxy sequencing work?

It uses fluorescently labeled ddNTPs, capillary electrophoresis, and computer analysis to determine DNA sequences rapidly.

What is shotgun sequencing?

A method that determines the sequence of small genomes by breaking them into overlapping fragments, sequencing them, and assembling the original sequence.

What is the principle behind Illumina sequencing?

It involves fragmenting DNA, amplifying it on a glass surface, and using reversible terminator nucleotides for sequencing by imaging.

What is the role of fluorescent markers in Illumina sequencing?

They indicate which base (A, T, C, or G) is added during DNA synthesis.

What is in situ hybridization used for?

To detect specific DNA or RNA sequences within cells, revealing when and where a gene is expressed.

What does a chromatogram represent in DNA sequencing?

It displays peaks corresponding to nucleotides, indicating the DNA sequence complementary to the original strand.

What is the significance of the 3' hydroxyl group in normal nucleotides?

It allows DNA polymerase to add another nucleotide, enabling continuous strand elongation.

What happens when a chain-terminating ddNTP is incorporated during DNA synthesis?

DNA strand elongation stops, creating fragments of varying lengths that can be analyzed for sequencing.

What is the outcome of PCR after several cycles?

The vast majority of DNA fragments correspond precisely to the target sequence defined by the primers.

What is the first step in the PCR process?

Heating the DNA to separate the strands.

What is the purpose of gel electrophoresis in PCR applications?

To analyze the amplified DNA fragments and confirm the presence of the target sequence.

What is the difference between genomic and cDNA clones?

Genomic clones represent the original DNA sequence including introns, while cDNA clones represent only expressed gene sequences (exons).

How does PCR contribute to diagnosing viral infections?

It allows for the detection of trace amounts of viral genetic material, providing a sensitive method for diagnosis.

What is the role of reverse transcriptase in PCR for RNA viruses?

It synthesizes cDNA from viral RNA, which can then be amplified by PCR.

What is the purpose of in situ hybridization?

To detect the location of viral DNA in tissues or cells, aiding in the identification of viral infections like HPV.

What is the first step in targeted gene replacement using embryonic stem (ES) cells?

Altering ES cells by introducing a DNA fragment containing an altered gene.

What is created when altered ES cells are injected into a mouse embryo?

A chimeric mouse that contains both normal and altered cells.

What is the outcome of breeding chimeric mice that carry the altered gene?

Transgenic 'knock-in' mice where both copies of the target gene are altered.

How do reporter genes function in gene expression studies?

They replace the coding sequence of a normal gene with that of a reporter gene, allowing visualization of gene expression patterns.

What is an example of a reporter gene used in studies?

Green fluorescent protein (GFP) or β-galactosidase.

How can scientists determine the role of regulatory sequences linked to a reporter gene?

By testing which cell types express the reporter when different combinations of regulatory sequences are used.

What is the function of the Cas9 protein in CRISPR-Cas9 genome editing?

It acts as molecular scissors to cut DNA at specific sites.

What guides the Cas9 protein to the target gene in CRISPR-Cas9?

A guide RNA (gRNA) engineered to match a specific DNA sequence.

What is the process of homologous recombination in CRISPR-Cas9?

The cell's repair machinery uses a donor DNA template to replace the original target gene with an altered version.

What is the purpose of creating transgenic plants using Agrobacterium?

To introduce engineered genes into plant cells for stable inheritance of desired traits.

What is the first step in the transgenic plant creation process?

Preparing leaf discs from a tobacco leaf.

What role does the selectable marker play in creating transgenic plants?

It allows for the selection of cells that have successfully integrated the recombinant DNA.

How are proteins mass-produced using expression vectors?

By inserting a gene into a plasmid with a strong promoter, driving high levels of mRNA transcription.

What is the significance of recombinant DNA technology in studying proteins?

It allows researchers to analyze proteins and their corresponding genes, facilitating studies on structure and function.

How can scientists move from protein to gene in recombinant DNA studies?

By analyzing a purified protein's sequence to identify the corresponding gene in a DNA database.

What is the reverse process of moving from gene to protein in recombinant DNA technology?

Inserting the protein-coding region of a gene into an expression vector for overexpression in host cells.

What is the overall benefit of recombinant DNA methods?

They allow scientists to study structure, function, and activity of genes and proteins by moving between them.