lecture 19 - recombinant DNA technology

Question 1

What is the key DNA structure used in recombinant DNA technology?

Answer 1

Plasmids

Question 2

What is a plasmid?

Answer 2

Usually circular pieces of dsDNA that can be altered to express specific genes.

Question 3

What are the 4 key components of a plasmid?

Answer 3

Ori (origin of replication), Selectable marker (usually antibiotic resistance gene), promotor, gene of interest

Question 4

What is the origin of replication of a plasmid?

Answer 4

Allows for the initiation of DNA replication using host DNA polymerase. This allows a large number of plasmids carrying the gene of interest to be produced within a single cell

Question 5

What is the selectable marker most commonly used in plasmids in recombinant DNA technology?

Answer 5

Antibiotic resistance genes

Question 6

What are plasmid antibiotic resistance genes?

Answer 6

Allows for the selection of cells contains the plasmid, as others will die when an antibiotic is introducd

Question 7

What is the promoter of a DNA recombinant plasmid?

Answer 7

Region that facilitates expression of the gene via transcription by providing a site for RNA polymerase to bind.

Question 8

What is found on either side of the gene of interest of a plasmid?

Answer 8

Restriction sites, so that the gene sequence can be transcripted properly

Question 9

What does the promoter region in a plasmid depend on?

Answer 9

The type of cell the plasmid will be in, to match the transcription factor machinery in that cell

Question 10

What is used to ‘cut and paste’ DNA into plasmids?

Answer 10

Restriction enzymes

Question 11

Where are restriction enzymes naturally found, and what is their function?

Answer 11

In bacteria as a defence mechanism to degrade foreign phage DNA - the enzymes will not cut methylated bacterial DNA but will cut unmethylated phage DNA

Question 12

Is phage DNA methylated or unmethylated?

Answer 12

Unmetheylated

Question 13

Is bacterial DNA methylated or unmethylated?

Answer 13

methylated

Question 14

What is used to ‘paste’ DNA into plasmids and repair the join?

Answer 14

DNA ligases

Question 15

What is the fucntion of DNA ligases in plasmid construction?

Answer 15

They allow DNA to be ‘pasted’ into plasmids using complementary base pairing. DNA ligase catalyses the formation of phopsphodiester bonds to the repair the nick in the backbone.

Question 16

What is transformation in terms of recombinant DNA technology?

Answer 16

The process of transferring edited plasmids into bacteria (or other cells) where they can be amplified

Question 17

How are transformed bacteria selected?

Answer 17

All cells are exposed to an antibiotic and only cells that contain the plasmid are able to grow/divide and form colonies

Question 18

What is the meaning of the ‘universal genetic code’?

Answer 18

All organisms ‘read’ the same codons/triplets as the same amino acids

Question 19

What additional process needs to occur so that prokaryotic cells can read eukaryotic DNA?

Answer 19

Prokaryotic genes have no introns, and so the cells cannot process eukaryotic introns. The eukaryotic coding sequence only must be used.