Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

Biotechnology Final > Recombinant DNA Technology > Flashcards

Recombinant DNA Technology Flashcards

1
Q

What is recombinant DNA technology?

A

Recombinant DNA technology is an umbrella term encompassing several protocols in which DNA is produced via artificial means.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What is recombinant DNA (rDNA)?

A

rDNA is DNA that is artificially created by combining DNA components from different organisms.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What disciplines lie under the umbrella of recombinant DNA technology?

A
  • Genetic engineering.
  • Gene cloning.
  • Molecular cloning.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

What are the two key enzymes used in recombinant DNA technology?

A
  • Restriction enzymes.
  • DNA ligase.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What is a vector in the context of recombinant DNA technology?

A

A vector is the vehicle that is used to transfer foreign genetic material into another cell (i.e., the host cell).

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What is a host cell in the context of recombinant DNA technology?

A

It is the living organism that takes up the rDNA to replicate it and sometimes express it.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

What is the first step of creating rDNA in the context of the source organism?

A

The DNA that carries the target gene of interest is isolated from the source organism and cut at specific sites using restriction enzymes.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

What happens to each enzymatically cut DNA fragments (i.e., insert DNA) of the source organism in the context of rDNA creation?

A

Each enzymatically cut DNA fragment (insert DNA) is joined (ligated) to a cloning vector (often a plasmid) using DNA ligase.

The vector has also been cut with the same restriction enzyme that has cut the source organism’s DNA, to ensure that the sticky ends match.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What affects the choice of vector in rDNA creation?

A
  • Size of DNA insert.
  • The type of host to be infected.
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What happens after we create a DNA construct (insert DNA ligated to a vector)?

A

We transfer it into a host cell, where it is maintained.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

How do we isolate the rDNA containing host cells after we create them?

A

By screening and identifying the host cell colonies of the rDNA molecules by identifying the specific colony containing the target gene.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

When were restriction enzymes first discovered?

A

In the 1960s, in bacteria which use these enzymes to defend against invading viral DNA.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What are restriction enzymes also called?

A

Restriction endonucleases.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What is the function of restriction endonucleases?

A

They cut DNA at specific palindromic sequences, creating either blunt or staggered cuts, depending on the endonuclease used.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

How are restriction endonucleases classified?

A

Into three classes based on their structure, specificity, and mechanism of action:
- Type I.
- Type II.
- Type III.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

How is the source DNA of the organism with the target gene and the vector DNA are prepared and joined together?

A

Both the source DNA and the vector need to be cut by the same restriction enzyme. Then, the DNA insert is ligated to the vector.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

What is a palindromic sequence?

A

A sequence that reads the same forward as backward.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

How are restriction endonucleases named?
Demonstrate it by showing how EcoRI is named.

A

Restriction endonucleases are named based on the bacterial species from which they were first isolated.
- Genus: Escherichia.
- Species: coli.
- Strain: R.
- Order Isolated: I.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

An example of a restriction endonuclease that creates sticky ends is…

A

EcoRI.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

An example of a restriction endonuclease that creates blunt ends is…

A

SmaI.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

When are sticky-ends used in rDNA technology?

A
  • When we need efficient and specific ligation.
  • When the orientation of the insert matters.
  • When we need to generate compatible sticky ends.
22
Q

When are blunt-ends used in rDNA technology?

A

Blunt ends can be ligated to any other blunt end regardless of the sequence. They are used when overhangs are undesirable.

23
Q

What is a disadvantage of blunt-ends in rDNA technology?

A

Ligation efficiency is lower than sticky-ends.

24
Q

What is a cloning vector?

A

A vector is a DNA molecule that is used to carry another foreign DNA molecule into the host cell, so it can be self-replicated and integrated into the host cell.

25
Q

What are the most common forms of vectors?

A
  • Plasmid (from bacterium).
  • A cell from the higher organism.
  • Viral DNA.
26
Q

What is the origin of replication (ori) in a cloning vector?

A

It is a DNA sequence within the vector that is recognized by the host cell’s replication machinery, enabling the vector (and any inserted DNA) to be replicated.

27
Q

Why do cloning vectors contain restriction sites?

A

They provide specific locations where the vector can be cut by restriction enzymes, allowing the insertion of the target (foreign) DNA.

28
Q

What is a selectable marker (often an antibiotic resistance gene) in a cloning vector?

A

It is a gene that allows researchers to identify and select cells that have successfully taken up the vector, usually by conferring resistance to a specific antibiotic.

29
Q

Why is a promoter included in a cloning vector?

A

It controls the transcription of the inserted gene, ensuring that the gene of interest is expressed under desired conditions in the host cell.

30
Q

What is a multiple cloning site (MCS)?

A

It is a short region containing numerous unique restriction sites, giving flexibility in choosing where and how to insert different DNA fragments.

31
Q

Why should a cloning vector be small in size?

A

A smaller vector is more easily introduced into host cells and is typically more stable during replication.

32
Q

Why do some vectors need to handle large DNA inserts?

A

Certain applications require cloning of larger genes or genomic fragments, so vectors must accommodate bigger pieces of foreign DNA.

33
Q

What are plasmid vectors and their characteristics?

A

Their insert size capacity is up to ~15 kb.

Their host organism is bacteria (commonly E. coli), and Yeast.

Their key features include:
- Circular DNA.
- High copy number.
- Easy to manipulate.

Applications:
- Cloning small DNA fragments.
- Gene expression studies.

Disadvantages:
- Not suitable for large DNA fragments.
- Relatively low transformation efficiency.

34
Q

What are bacteriophage vectors and their characteristics?

A

Their insert size capacity is 9-23 kb.

Their host organism is bacteria (E. coli).

Their key features include:
- Linear DNA.
- Efficiently packaged into phage particles.
- Infection-based introduction into host cells.

Applications:
- cDNA and genomic library construction.
- High-efficiency cloning.

Disadvantages:
- Limited insert size.
- Complex cloning steps.

35
Q

What are cosmid vectors and their characteristics?

A

Their insert size capacity is 35-45 kb.

Their host organism is bacteria (E. coli).

Their key features include:
- Hybrid of plasmid and phage features.
- Contain “cos” sites for packaging into phage particles.
- Replicate as plasmids in host cells.

Applications:
- Cloning medium-sized DNA fragments.
- Genomic library construction.

Disadvantages:
- Instability with larger DNA inserts.

36
Q

What are BAC (bacterial artificial chromosomes) vectors and their characteristics?

A

Their insert size capacity is 100-300 kb.

Their host organism is bacteria (E. coli).

Their key features include:
- Based on the F-plasmid origin.
- Low copy number.
- Stable maintenance of large inserts.

Applications:
- Cloning large DNA fragments.
- Genomic library construction.
- Genome mapping.

Disadvantages:
- Low copy number complicates DNA yield and manipulation.

37
Q

What are YAC (yeast artificial chromosomes) vectors and their characteristics?

A

Their insert size capacity is 200-2,000 kb.

Their host organism is yeast.

Their key features include:
- Linear DNA with telomeres and centromere.
- Contains a yeast origin of replication.
- Can accommodate very large DNA fragments.

Applications:
- Cloning very large DNA fragments.
- Physical mapping.
- Genome sequencing projects.

Disadvantages:
- High propensity for recombination.
- Insert instability.

38
Q

What are HAC (human artificial chromosomes) vectors and their characteristics?

A

Their insert size capacity is up to several megabases (Mbs).

Their host organism is human cells.

Their key features include:
- Contain a human centromere, telomeres, and replication origins.
- Behave like a natural chromosome.
- Maintained independently in host cells.

Applications:
- Gene therapy vectors.
- Functional studies in human cells.
- Expression of large genes or gene clusters.

Disadvantages:
- Technical challenges in construction.
- Difficulty with cell delivery and stable maintenance.

39
Q

What are some examples of host cells used in rDNA technology?

A
  • Prokaryotes (E. coli).
  • Yeasts.
  • Insects.
  • Plants.
  • Mammalian cells.
  • Transgenic animals.
40
Q

What are the ways the foreign genetic material is inserted into host cells?

A
  • Transfection: non-viral DNA into eukaryotic cells.
  • Transformation: uptake of foreign DNA by bacteria.
  • Transduction: virus-mediated transfer of DNA between bacteria.
41
Q

What is gene optimization in therapeutic protein expression?

A

It involves modifying the DNA sequence (e.g., using host-preferred codons, removing introns) to enhance protein production and stability in the host.

42
Q

How is host engineering used to improve protein yields?

A

By using or creating host strains that lack specific proteases or that are genetically modified to minimize protein degradation and boost expression levels.

43
Q

What does PTM engineering focus on?

A

Adjusting PTMs (e.g., glycosylation, phosphorylation) by engineering yeast or mammalian cells and co-expressing enzymes like kinases or phosphatases.

44
Q

Why is process scale-up and bioreactor optimization important?

A

It ensures controlled conditions (pH, temperature, oxygen, nutrients) at larger scales to maintain high yields and consistent product quality.

45
Q

What does efficiency downstream processing entail?

A

It uses effective purification methods to isolate and purify the therapeutic protein with minimal loss and high overall quality.

46
Q

How was insulin originally obtained?

A

It was extracted from the pancreases of animals, such as pigs, before rDNA methods became available.

47
Q

How is recombinant insulin produced today?

A

Through rDNA technology: the human insulin gene is inserted into a bacterial host (e.g., E. coli) to produce insulin in large fermentation tanks.

48
Q

Why is recombinant insulin advantageous over animal-derived insulin?

A

It is more consistent in quality, reduces the risk of immunogenic reactions, and can be produced n larger, more reliable quantities.

49
Q

Why is hGH crucial for the human body?

A

It plays a key role in growth, development, and metabolism; its deficiency leads to conditions such as hypopituitarism.

50
Q

How was hGh historically obtained, and what was the major risk?

A

It was extracted from cadaveric pituitary glands, which led to cases of Creutzfeldt-Jakob disease when contaminated material was used in 1985.

51
Q

How is recombinant hGH produced safely today?

A

By cloning and expressing the hGH gene in mammalian cells (e.g., Chinese hamster ovary cells), ensuring high yields of pure, uncontaminated hormone.

52
Q

Why are mammalian cells preferred for hGH production?

A

They provide proper PTMs like glycosylation, which is necessary for the correct folding and function of hGH.

Biotechnology Final (2 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2025 Bold Learning Solutions. Terms and Conditions