Shuttle Vectors and Real-Time PCR

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Questions and Answers

What is the primary purpose of shuttle vectors in genetic modification?

To synthesize RNA from DNA templates.

To increase the size of plasmids for cloning.

To transport cloned DNA sequences among different organisms. (correct)

To amplify entire genomes efficiently.

Which enzyme is used in reverse-transcription PCR (RT-PCR)?

DNA ligase

RNA polymerase

Reverse transcriptase (correct)

DNA polymerase

What significant limitation does PCR have in terms of DNA amplification?

It cannot amplify non-coding DNA sequences.

It can only amplify relatively small specific sequences of DNA. (correct)

It can only amplify mitochondrial DNA.

It is limited to prokaryotic DNA amplification.

What is the function of the fluorescent dye in quantitative PCR (qPCR)?

To measure the levels of amplification during PCR cycles. Signup and view all the answers

Which of the following vectors can typically accept larger pieces of DNA for cloning?

Viral DNA vectors Signup and view all the answers

In PCR, what component is NOT added during the preparation stage?

Reverse transcriptase Signup and view all the answers

Which type of virus is commonly used as a vector for gene therapy due to its ability to accept large DNA fragments?

Retrovirus Signup and view all the answers

What critical factor influences the choice of a suitable vector for cloning?

The type of foreign DNA and the organism receiving the gene Signup and view all the answers

What is the purpose of treating the filter with detergent (SDS) in the colony hybridization process?

To lyse bacteria and release their contents. Signup and view all the answers

Which of the following statements about DNA probes is true?

DNA probes are synthesized to be complementary to a target gene. Signup and view all the answers

What color indicates the presence of an intact lacZ gene when bacteria hydrolyze X-gal?

Blue Signup and view all the answers

What is a common challenge faced after isolating white colonies during cloning?

Identifying whether the colonies contain the desired fragment of foreign DNA. Signup and view all the answers

How is the presence of a matched DNA probe detected during the hybridization process?

Through the use of an enzyme or fluorescent dye label. Signup and view all the answers

What follows the process of filtering the colonies on nitrocellulose?

Growing the bacterial isolates in culture. Signup and view all the answers

In colony hybridization, what is the consequence of the DNA probe failing to find a match?

The target gene is confirmed absent from the colony. Signup and view all the answers

What component is necessary for identifying pathogens using DNA chips?

A series of DNA probes complementary to target sequences. Signup and view all the answers

What role does ampicillin play in the blue-white screening procedure?

It prevents the growth of bacteria that do not take up the plasmid. Signup and view all the answers

Which enzyme does the lacZ gene encode for in the blue-white screening process?

Beta-galactosidase Signup and view all the answers

In the context of the blue-white screening, what happens to bacteria that successfully uptake the plasmid containing the lacZ gene?

They produce blue colonies on X-gal medium. Signup and view all the answers

What is the function of X-gal in the culture medium?

To serve as a colorimetric substrate for beta-galactosidase. Signup and view all the answers

What occurs when foreign DNA is introduced into the plasmid during the blue-white screening?

The lacZ gene is disrupted, leading to white colonies. Signup and view all the answers

What type of genetic element is the lacZ gene considered?

A selectable marker for bacterial culture. Signup and view all the answers

What is the primary purpose of using a restriction enzyme in the blue-white screening procedure?

To cut both plasmid DNA and foreign DNA at specific sites for ligation. Signup and view all the answers

Which statement accurately describes the expected outcome of a successful transformation in the blue-white screening?

Bacteria will either produce white or blue colonies depending on the uptake of foreign DNA. Signup and view all the answers

Which of the following substances is used to break down mucous secretions in cystic fibrosis patients?

Pulmozyme (rhDNase) Signup and view all the answers

What type of vaccine is developed using the Canarypox virus carrying virus genes?

Feline Leukemia Vaccine Signup and view all the answers

Which cell type is used to produce the monoclonal antibody Orthoclone OKT3?

Mouse Cells Signup and view all the answers

What role does Tumor Necrosis Factor (TNF) play in cancer treatment?

Destroys tumor cells Signup and view all the answers

Which of the following vaccines is NOT produced using E.coli?

Muromonab-CD3 Signup and view all the answers

How are small interfering RNAs (siRNAs) formed in the gene silencing process?

Through processing by the enzyme Dicer Signup and view all the answers

What therapeutic use does the enzyme Superoxide Dismutase (SOD) have?

Minimizes damage from oxygen free radicals Signup and view all the answers

Which of the following best describes gene silencing?

A natural defense against viruses Signup and view all the answers

What is the primary purpose of the micropipette in the microinjection process?

To precisely inject copies of the gene into the nucleus Signup and view all the answers

What is a major difference between a plasmid library and a phage library?

Plasmid libraries are derived from bacterial cells, while phage libraries are derived from viruses. Signup and view all the answers

Why is cDNA considered a common method for obtaining eukaryotic genes?

It is less dependent on the presence of introns in eukaryotic genes. Signup and view all the answers

What is the function of reverse transcriptase in the cDNA method?

To create a DNA copy of mRNA Signup and view all the answers

What steps follow the action of reverse transcriptase in the cDNA synthesis process?

mRNA is enzymatically digested and then a complementary strand of DNA is synthesized Signup and view all the answers

What problem is specifically associated with using the cDNA method?

The presence of excess intron-derived RNA leads to complications. Signup and view all the answers

What is the role of processing enzymes in the nucleus during RNA transcription?

To remove introns and splice exons together Signup and view all the answers

Why might microinjection be considered impractical for bacterial and fungal cells?

Bacterial and fungal cells lack nuclei. Signup and view all the answers

Study Notes

Shuttle Vectors and PCR Techniques

Shuttle vectors facilitate gene transfer across various organisms, including bacterial, yeast, and mammalian cells.
Real-time PCR (qPCR) quantifies DNA by incorporating fluorescent dyes, allowing measurement of fluorescence after each cycle.
Reverse-transcription PCR (RT-PCR) employs viral RNA or cellular mRNA as a template to generate DNA via reverse transcriptase, followed by amplification.
Viral DNA vectors can accommodate larger DNA inserts compared to plasmids, enabling cloning within host cells.
Primers define the specific sequences targeted during PCR; however, PCR amplifies only relatively small DNA fragments and cannot target entire genomes.

Types of Genes and Libraries

cDNA libraries are created from RNA templates, where mRNA undergoes transcription to produce complementary DNA (cDNA).
The process involves splicing introns from exons and subsequently using DNA polymerase to synthesize a double-stranded piece of DNA.
Blue-white screening leverages the lacZ gene for identifying bacteria with plasmids containing foreign DNA, distinguishing colonies based on color.

Colony Hybridization and DNA Probes

Colony hybridization utilizes DNA probes that are complementary to target genes; successful binding indicates the presence of a specific cloned gene.
DNA chips with arrays of probes are used for pathogen identification by matching probes to target DNA sequences.

Biotechnology Applications

Therapeutics like interleukins, monoclonal antibodies, and enzymes (e.g., Pulmozyme) are produced using recombinant DNA technology in various host organisms.
Veterinary vaccines use attenuated viruses carrying genes from pathogens, exemplified by canine distemper and feline leukemia vaccines.

Gene Silencing and Genome Projects

Gene silencing, a natural defense mechanism, uses small interfering RNAs (siRNAs) to target and degrade specific mRNAs, similar to miRNA functions.
The first complete genome sequenced was from a bacteriophage in 1977, marking a significant milestone in genomic research.

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Description

This quiz covers the critical role of shuttle vectors in molecular biology, particularly in the context of real-time PCR (qPCR). Explore how these vectors facilitate the transfer of DNA sequences across various organisms, enabling effective measurement and analysis of fluorescence during PCR cycles.