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Ocular Health & Disease > 8. Gene cloning > Flashcards

8. Gene cloning Flashcards

1
Q

What is recombinant DNA technology?

A

A set of procedures to join together segments of DNA.

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2
Q

How to make alot of copies of the recombinant DNA?

A

Put recombinant DNA in a bacterial cell.

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3
Q

What are the basic steps in gene cloning?

A
  1. Purification of DNA
  2. Restriction endonucleases cut DNA
  3. Insert target DNA into a vector to produce recombinant DNA molecule
  4. Vectors used to transport recombinant DNA into other living cells that multiply quickly
  5. Growth to give large number of copies
  6. Identification of the cells containing recombinant DNA
  7. Idenitfication of cells containing gene of interest
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4
Q

What are plasmids?

A

Circular extra chromosomal pieces of DNA.
They multiply independently of the bacterial chromosome

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5
Q

Plasmids carry genes that give a useful characteristic, give an example of a useful characteristic

A

antibiotic resistance

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6
Q

What is bacteriophage?

A

Viruses that infect bacteria

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7
Q

What does the life cycle of a bacteriophage involve?

A
  1. Attachment of the virus to bacterial cell receptors and injects DNA into cells.
  2. Replication of DNA
  3. New virus particles and release from bacteria cell
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8
Q

What are restriction endonucleases?

A

Enzymes that cut double stranded DNA breaking phosphodiester bonds.

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9
Q

Which type of restriction endonuclease is important for cloning?

A

Type II

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10
Q

What kind of sequences do restriction endonucleases break?

A
  1. They break specific sequences usually palindromic
  2. 4,5,6 bases long.
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11
Q

What is purification of DNA?

A

Removing proteins, lipids which could interfere with the gene cloning.

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12
Q

How is DNA purified?

A

Using enzymes and chemical treatments.

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13
Q

Why do we need to insert target DNA into a vector?

A

Can’t put target DNA directly into a cell as it would be unstable and be broken down.

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14
Q

What are the 2 most common vectors for gene cloning?

A

Plasmids and bacteriophage DNA

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15
Q

What is used to make the phosphodiester bond?

A

ligase enzyme

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16
Q

What are the ways to inserting recombinant DNA into host?

A
  1. Transformation
  2. Transfection
  3. Micro-injection
  4. Electroporation
17
Q

What is electroporation?

A

High voltage impulses destablises the cell membrane of the bacterium so DNA can enter from solution

18
Q

Why do we need recombinant DNA for humans?

A

Some disease are caused by a lack of protein, so we need proteins that can be extracted from blood but this is expensive and you only gather small amounts.

19
Q

What are some other applications of recombinant DNA?

A
  1. Agricultural - improve crops, resistance to pest damage, safety of GM foods
  2. Environmental - saving endangered species
  3. Forensic medicince - DNA fingerprinting compare.

Ocular Health & Disease (6 decks)

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