Lecture 2

Question 1

Gene cloning is a biological method of

Answer 1

Purification

Question 2

Where does the gene of interest get inserted into

Answer 2

Vector

Question 3

Give an example of a vector

Answer 3

Plasmid

Question 4

Once placed into a vector where does it go next

Answer 4

Introduced into a host cell

Question 5

When the vector replicates what does this allow

Answer 5

Allows large amounts of it to be isolated and used for multiple applications

Question 6

Once a gene has been cloned what can be determined

Answer 6

Nucleotide sequence

Question 7

How is a clone used to determine where the cloned gene is expressed

Answer 7

It is used as a probe

Question 8

Why would you use a clone as a probe

Answer 8

Determine where and when the clones gene is expressed

Question 9

How is gene function determined

Answer 9

Over expression or mutation of the cloned gene

Question 10

What is the gene product

Answer 10

Protein

Question 11

Gene product can be purified for what

Answer 11

Enzymes
Antibodies
Protein structure

Question 12

What is transgenesis

Answer 12

Genes that are artificially expressed in other organisms

Question 13

Name 6 things you would need in a gene cloning toolkit

Answer 13

Source of DNA

Polymerase chain reaction

Restriction endonucleases

DNA Ligase

Vector

Host cells

Question 14

Source of DNA will include

Answer 14

Genomic DNA gDNA

Complementary DNA cDNA

Question 15

Where does cDNA derive from

Answer 15

Reverse transcription of mRNA

Question 16

What is a clone

Answer 16

DNA molecule that is identical to the DNA molecule from where it derived from

Question 17

Does the mRNA have any introns

Answer 17

No

Question 18

When can we only run pcr

Answer 18

Nucleotide sequence is known

Question 19

Why is it good to use pcr

Answer 19

Produce lots of the desired DNA sequence

Question 20

What is restriction endonucleases for

Answer 20

To cut DNA

Question 21

Restriction endonucleases recognise what

Answer 21

4bp frequent
6bp intermediate
8bp infrequent

Question 22

What is DNA Ligase for

Answer 22

Join together fragments

Question 23

What is a vector

Answer 23

Where cloned DNA is inserted

Question 24

What are the two ways of thinking of a gene clone

Answer 24

Method trying to identify a gene in the first place

As a way of manipulating the gene once it has been identified

Question 25

What are the 3 approached for gene identification

Answer 25

Mapping Homology Functional complementation of mutants

Question 26

What is a collection of gDNA fragments

Answer 26

gDNA library

Question 27

How to make a gDNA library

Answer 27

Digest gDNA with restriction enzymes and Ligase it into a vector cut with the same enzyme

Question 28

What are the 2 approaches for creating a gDNA

Answer 28

Complete digest and partial digest

Question 29

What is complete digest

Answer 29

Is with one or several enzymes, incase the chosen gene contains a restriction site for the enzymes used

Question 30

What is partial digest

Answer 30

Is with frequent cutting enzyme. This creates overlapping of fragments.

Question 31

After the plasmids are inserted into the host what needs to happen next

Answer 31

Select the mutants with the plasmids Screen for hosts that can grow on the medium lacking arginine Isolate plasmid from the colony Sequence the DNA fragment

Question 32

When a plasmid has a fragment inserted what is this called

Answer 32

Recombinant DNA

Question 33

What do we use the library for

Answer 33

Isolate genes Identify a full sequence Identify a close related species

Question 34

How can we get a probe

Answer 34

It could be a fragment of the target gene Or it could be a gene from a different species that share the same gene

Question 35

After gene manipulation what do you do next

Answer 35

Analyse the cloned gene sequence Analyse gene expression profile Analyse gene function Produce recombinant protein Make transgenic organisms

Question 36

Why do we analyse the gene expression profile

Answer 36

Determine where and when the gene is expressed

Question 37

Why do we analyse gene function

Answer 37

Examine phenotype Over express the gene Mutation in the genes Associate it with certain diseases

Question 38

Why do we produce recombinant proteins

Answer 38

Raise antibodies for gene products

Question 39

Why do we make transgenic organisms

Answer 39

So we can express the gene to give some desired trait

Question 40

What do pcr primers do

Answer 40

Used to incorporate restriction enzyme sites into the gene

Question 41

What must be used to produce a recombinant protein

Answer 41

Expression vector

Question 42

What does pcr do

Answer 42

Amplifies a particular ba sequence for cloning