Genetic Engineering - Lecture 21

Question 1

What are the two types of gene libraries?

Answer 1

○ cDNA
○ Genomic

Question 2

What does molecular cloning allow?

Answer 2

○ Reduces the complexity of DNA which allows genes to be studied
○ Allows large scale production and analysis of purified single sequences

Question 3

How is DNA cut and spliced?

Answer 3

○ Restriction endonuclease which cuts DNA that are palindromic sequences
○ Produces sticky ends (single strand overhang) which can be rejoined
○ DNA fragment can be inserted where that cut had been made as long as its been cut with the same restriction endonuclease

Question 4

What are the outcomes of a cut plasmid and cut DNA fragment?

Answer 4

○ Unmodified plasmid
○ Recombinant plasmid
○ Circularised DNA fragment (will not replicate)

Question 5

How is the unmodified plasmid prevented?

Answer 5

Phosphatase removes 5’ phosphatase which prevents the ends of plasmid coming together

Question 6

What does the genomic library include?

Answer 6

○ Contains all of the gene
○ Species-specific

Question 7

What does the cDNA library include?

Answer 7

○ Only expressed genes (mRNA)
○ Species and tissue specific
○ Must be converted to DNA to be cloned
○ Converted using reverse transcriptase

Question 8

What are the features of cDNA library?

Answer 8

○ Comprises expressed genes (transcriptome)
○ ‘Libraries’ from different tissues contain different sequences
○ No untranscribed sequences

Question 9

What are the features of genomic library?

Answer 9

○ Comprises sequences representing the genome - same in all tissues
○ Includes introns and regulatory sequences as well as exons
○ “Raw material” for gene mapping and genome projects

Question 10

What is general principle of making therapeutic proteins

Answer 10

○ Fuse coding region of gene to a strong promoter
○ Insert recombinant gene into host
○ Host multiplies and overproduces proteins
○ Isolate and purify proteins

Question 11

What are the advantages of using bacteria as a host system?

Answer 11

○ Cheap
○ Fast-growing
○ Easy to maintain
○ Stable

Question 12

What are the disadvantages of using bacteria as a host system?

Answer 12

○ Proteins may be insoluble/denatured
○ No post-translational modification

Question 13

What are the advantages of using animal cells as a host system?

Answer 13

○ Post-translational modification
○ Soluble/properly folded

Question 14

What are the disadvantages of using animal cells as a host system?

Answer 14

○ Expensive
○ Unstable

Question 15

How is insulin synthesised in bacteria?

Answer 15

Synthesise 2 chains apart then join in a particular chemical environment