Module 10 - Genetic engineering

Question 1

Molecular cloning

Answer 1

The cutting, joining, and propagating of recombinant DNA

Question 2

What does molecular cloning do

Answer 2

Reduces DNA complexity

Allows large-scale production and analysis of purified single sequences

Question 3

Steps in molecular cloning

Answer 3

1 - Isolate DNA
2 - Cut DNA (usually through restriction endonucleases)
3 - Join to a “vector” recombinant
4 - Introduce vector into bacteria (transformation)
5 - DNA is amplified as bacteria grows

Question 4

Restriction endonucleases: what do they do?

Answer 4

Cut at palindromic sequences, forming ‘sticky ends’ which can be rejoined

Many restriction endonucleases, each with different sequence specificities

Question 5

Three possibilities through trying to introduce a DNA fragment to a cut plasmid

Answer 5

Unmodified plasmid (most likely) - the sticky ends joined up and the DNA fragment is not introduced (recircularisation)

Circularised DNA fragment - DNA fragment binds to itself without binding to the plasmid

Recombinant plasmid - DNA fragment is integrated

Question 6

How do we prevent unmodified plasmid formation?

Answer 6

Treating the cut plasmid with a phosphatase which snips off the 5’ phosphate from the plasmid, preventing ‘sticky end’ bonding

Question 7

The process of cultivating bacteria with recombinant plasmids

Answer 7

Bacteria put in antibiotics, those with antibiotic resistance (recombinant plasmid) will survive and grow

Question 8

DNA libraries: what two places can they be obtained from?

Answer 8

Nuclear DNA

mRNA

Question 9

Nuclear DNA DNA library

Answer 9

Contains all genes (genomic library), is the main source of gene library formation

Question 10

mRNA cDNA library

Answer 10

It uses expressed mRNA which has been converted to DNA by reverse transcriptase

It is usually cell-type (tissue) specific

Used mainly for monitoring protein expression and isolating full reading frames

Question 11

Formation of DNA libraries (cDNA library)

Answer 11

The mRNA is isolated, reverse transcriptase converts mRNA to cDNA, inserted into a vector and transformation occurs, on average, only one sequence is taken in by colony (1 colony - 1 cDNA library)

Question 12

Formation of DNA libraries (genomic library)

Answer 12

DNA is isolated and then cut using restriction nucleases (~50kb strands produced), each stand inserted into a vector, and transformation occurs, on average only one sequence is taken by colony (1 colony - 1 strand from the genomic library)

Question 13

cDNA library: what is it, how is it made, and what is a key part to it?

Answer 13

A library of all the transcripted genes (transcriptome)

Using reverse transcriptase on mRNA

It is specific to each tissue type

Question 14

DNA library: what is it, how is it made, and what is a key part to it?

Answer 14

A library of the entire genome

Isolating the DNA

It is specific to the species it’s from

Question 15

Therapeutic uses of genetic engineering

Answer 15

Can mass produce useful proteins that have various therapeutic uses

Question 16

Advantages of using bacteria cells as host systems for mass-producing proteins

Answer 16

Cheap, grow fast, easy to maintain, and stable

Question 17

Disadvantages of using bacteria cells as host systems for mass-producing proteins

Answer 17

Proteins may be insoluble or denatured

No post-translational modification

Question 18

Advantages of using animal cells as host systems for mass-producing proteins

Answer 18

Can do post-translational modification

Proteins are soluble and properly folded

Question 19

Disadvantages of using animal cells as host systems for mass-producing proteins

Answer 19

Expensive, unstable