Lecture 24: Recombinant DNA I

This set of flashcards covers key concepts and techniques related to recombinant DNA technology discussed in Lecture 24.

What are vectors in recombinant DNA technology?

Carriers for pieces of DNA.

What are the two broad categories of vectors?

Viral (bacteriophage & virus) and plasmids.

What is a plasmid?

An independently replicating, small, extra-chromosomal piece of DNA.

What role do restriction endonucleases play in DNA manipulation?

They cleave both strands of a double-stranded DNA molecule at specific sites.

What is the purpose of ligase in recombinant DNA technology?

To seal nicks in the DNA backbone.

What happens during the lytic cycle of bacteriophage Lambda?

The phage attaches to the host cell, injects DNA, replicates, and causes cell lysis to release progeny phage.

What is the function of terminase enzyme?

To cut concatomer into single copies of the genome.

What happens if the amount of DNA inserted into the Lambda phage genome is less than 75%?

The phage will not package the DNA and won't pair with the host.

What is the genome size limit for Lambda phage as a vector?

Approximately 48 kb, with optimal packing between 75% and 110%.

What is an adenovirus?

A double-stranded DNA virus used as a vector for eukaryotic cells.

What is a selectable marker in plasmids?

A gene that allows for the identification of cells that contain the plasmid, often antibiotic resistance.

What is the purpose of a multiple cloning site (mcs) in plasmids?

To provide a location for the introduction of foreign DNA fragments.

What is a blue-white screening?

A method to identify successful gene incorporation using the lacZ gene.

How do restriction enzymes generate sticky ends?

By cleaving DNA at specific sequences, creating overhangs that can base pair with compatible ends.

What is the function of DNA phosphatase?

To remove 5' phosphates from DNA to prevent self-ligation.

What is directional cloning?

A method that allows DNA to be inserted in a specific direction into a vector.

How do blunt-end ligations differ from sticky-end ligations?

Blunt-end ligations do not have overhangs and can’t control insert orientation.

What is required for ligase to seal nicks in DNA?

A 5' α-phosphate and a 3' hydroxyl.

What can happen during self-ligation of DNA?

DNA can repair itself instead of binding with other DNA, defeating the cloning purpose.

What is the main goal of using adenoviral vectors in gene therapy?

To introduce DNA into cells without producing progeny virus.

How can adenoviral vectors be modified?

By changing their external membrane to target specific cell types.

What is a concatomer?

A long continuous strand of repeats of the same DNA sequence.

What is the significance of generating incompatible ends in DNA cloning?

It prevents self-ligation and allows specific DNA insertion.

What does T4 DNA kinase do?

It adds a phosphate group to the 5' end of DNA.

What are the functions of reverse transcriptase?

To synthesize DNA from an RNA template.

What is the purpose of using a high-copy plasmid vector?

To ensure a larger number of copies of the inserted DNA.

What types of cells can adenoviral vectors infect?

Only certain types of cells as targeted by design.

What does the term 'self-ligation' refer to?

The ability of DNA ends to join with themselves instead of with other DNA.

What is the process of theta replication?

A method of DNA replication typically used by circular DNA.

How can a plasmid selection marker work?

By allowing only cells with the plasmid to survive in the presence of an antibiotic.

What are sticky ends?

Overhangs created after the cutting of DNA that can base pair with other compatible sequences.

What can help prevent self-ligation in DNA constructs?

The removal of 5' phosphates using phosphatase.

What is a potential drawback of blunt-end cloning?

Lack of control over insert orientation and compatibility.

What is generation of blunt ends?

The process of creating straight cuts in DNA that lack overhangs.

What is the role of ligase in constructing recombinant DNA?

To join DNA fragments together by sealing nicks.

Why is it important to prevent self-ligation?

To ensure that inserted DNA can pair with plasmid DNA instead of repairing itself.

What is a phage?

A virus that infects and replicates within bacteria.

Why might certain genes be removed from adenoviral vectors?

To allow space for additional DNA without compromising functionality.

What happens when the vector is overloaded with DNA?

The capsid cannot package the DNA properly.

What does the term 'palindrome' refer to in genetic sequences?

A DNA sequence that reads the same forward and backward.

What is the typical size of a useful plasmid vector?

About 3 kb.

How does thermal motion contribute to DNA ligation?

It helps brought together sticky ends of DNA fragments for ligation.

What is the purpose of creating 'sticky ends' with restriction enzymes?

To enable the joining of different DNA fragments through base pairing.

What happens when a cell with a plasmid encounters an antibiotic?

The cells containing the plasmid with antibiotic resistance will survive.

What is the concept of gene libraries in recombinant DNA technology?

A collection of cloned DNA fragments that represents the genome of an organism.