L5-6 Recombinant DNA Technology Flashcards

Understand the principles and potential applications of recombinant DNA technologies.

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1
Q

Recombinant DNA is when two pieces of DNA are…

A

ligated artificially to perform a modified function

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2
Q

The resources required to create recombinant DNA are (4)…

A
  1. Enzymes
  2. Vector
  3. Cells
  4. DNA or RNA raw material
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3
Q

Four groups of enzymes are used in the production of recombinant DNA…

A
  1. Restriction endonuclease (site-specific cleavage)
  2. DNA ligase (the ‘glue’)
  3. TAQ polymerase (used in PCR)
  4. Reverse transcriptase (converts RNA back to DNA)
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4
Q

Restriction endonucleases originated as bacterial self-defence mechanisms. They tend to recognise what sort of sequences?

A

4-8 base pair palindromic sequences

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5
Q

Clevage can be symmetrical (resulting in blunt ends), or…

A

Asymmetrical (resulting in ‘sticky ends’)

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6
Q

Vectors are a means of introducing exogenous DNA into…

A

Host cells

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7
Q

Plasmids are commonly used as…

A

Vectors

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8
Q

Vectors other than plasmids include (2)…

A

Bactereophages and Cosmid/phagemids

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9
Q

Specially made plasmids often have a variety of ? to ensure compatibility with a variety of restriction enzymes

A

Restriction sites (known as a Multiple Cloning Site, MCS, or a polylinker)

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10
Q

A specific method for isolating the gene we want to insert involves…

A

Separating strands, attaching primers and amplifying with PCR

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11
Q

There are two ‘random’ ways to isolate the required gene, these are…

A
  1. Isolate the DNA, cleave, leaving random fragments

2. Isolate the mRNA, use reverse transcriptase, producing a cDNA library

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12
Q

cDNA stands for…

A

Complementary DNA

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13
Q

In order to introduce a vector into the cell, it must be made…

A

Competent

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14
Q

A cell may be made competent via…

A

🔥 Heat shock + CaCl

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15
Q

Adding exogenous DNA to the cell is known as…

A

Transformation

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16
Q

A further way to check if the cells cultured contain the recombinant DNA is to…

A

Use a hybridisation probe (ssDNA) for the sequence of interest (Southern blot)

17
Q

CFU stands for…

A

Colony-Forming Unit

18
Q

For a recombinant gene to successfully expressed as a recombinant protein, it must contain a…

A

Promoter recognised by the cell’s RNA polymerase

19
Q

How do we ensure the insert is joined to the plasmid in the correct orientation?🔁

A

Use a different restriction endonuclease for each end

plasmid—-⧑ new gene ◑—plasmid

20
Q

Define: intron

A

A non-coding region of DNA removed during maturation of mRNA by spliceosomes

21
Q

Reverse transcriptase is used to obtain cDNA from mRNA, rather than using spliceosomes, because…

A

Splicesomes are complex and difficult to assemble/utilise

22
Q

We can choose from four main Expression Systems to express our recombinant DNA into a protein (4)…

A
  1. Bacterial
  2. Yeast
  3. Insect
  4. Mammalian
23
Q

Insect cells are a good alternative to mammalian cells as (5)…

A

They are capable of high-level protein expression

Correct folding of mammalian proteins

Post-translational modification

Cheaper than mammalian cells

Fewer ethical issues