Chapter 9-Recombinant DNA

Question 1

What is recombinant DNA?

Answer 1

• genetic engineering of new combinations of DNA segments that are not found naturally

Question 2

Applications of recombinant DNA

Answer 2

1. Basic research, such as analysis of gene function and protein expression, purification and detection
2. Production of therapeutic antibodies and vaccines
3. Diagnostics and treatment of diseases
4. Genetic manipulation of crops

Question 3

What are vectors?

Answer 3

• double stranded and often circular DNA molecule used a s a vehicle to transfer foreign DNA into another cell
• cloning vectors (solely used for cloning and replication) and expression vectors (used to express protein of interest)

Question 4

Essential features of plasmids for cloning

Answer 4

1. Origin of replication
2. Selectable markers
3. Promoter
   4 multiple cloning site

Question 5

Function of selectable markers

Answer 5

• permits selection of host cells that carry the recombinant plasmid (successfully transformed)

Question 6

What is the MCS?

Answer 6

• contains a set of restriction sites that allow for insertion of foreign DNA
• foreign DNA needs to be ligated into the vector ‘in frame’ with the ATG start codon such that the upstream promoter drives transcription of cDNA

Question 7

How does ampicillin kill bacteria?

Answer 7

• inhibits action of the bacterial enzyme transpeptidase that is require to make the bacterial cell wall

Question 8

How does neomycin kill bacteria?

Answer 8

• binds to 30s and 16s ribosomes, inhibiting protein synthesis and stopping bacterial growth

Question 9

How does tetracycline kill bacteria?

Answer 9

• inhibits protein synthesis by blocking tRNA insertion into the bacterial ribosome

Question 10

What does B-galactosidase do to X-Gal?

Answer 10

X gal turns blue

Question 11

Features of primers

Answer 11

1. 15-25 nucleotides long
2. Tm of 50-60 degrees celsius
3. Tm of forward and reverse primers should be similar
4. Primer sequence given in 5’ to 3’ direction

Question 12

What is epitope tagging used for?

Answer 12

1. Protein purification
2. Visualisation by fluorescence microscopy
3. Detection by WB

Question 13

Why do we clone fusion (tagged) proteins?

Answer 13

1. Protein isolation by affinity purification
2. Detection of proteins in cells by direct fluorescence microscopy
3. Detection of proteins in cells by indirect immunofluorescent microscopy

Question 14

Transfection methods for mammalian cells

Answer 14

1. Micro injection
2. Lipofection
3. Electroporation
4. Gene gun

Question 15

How to approximate Tm

Answer 15

Tm= 2(A+T) + 4(G+C)