Cloning & genetic libraries

Question 1

What is cloning?

Answer 1

Copying ____ to produce multiple identical copies:

• organism
• plant
• cell culture
• piece of DNA

Question 2

Which enzymes are used during cloning?

Answer 2

Restriction endonucleases, DNA pol., DNA ligase, reverse transcriptase, phosphatase

Question 3

How is an insert prepared for cloning?

Answer 3

a) restriction endonuclease digestion: cut @ specific sites
b) physical fragmentation: then use DNA pol. to fill sticky ends => blunt ends
c) making complimentary copy (ds-cDNA) of RNA using reverse transcriptase: mRNA -> ss-cDNA -> ds-cDNA
d) amplify specific regions w/ PCR

Question 4

How is a vector prepared for cloning?

Answer 4

1. digested w/ restriction enzyme => sticky ends that complements DNA to be cloned
2. (OPTIONAL) dephosphorylate termini of vector using phosphate enzyme = prevent recircular w/out an insert
3. vector is purified = avoid reagents interering w/ ligase react.

Question 5

List essential elements of a cloning vector

Answer 5

• Multiple cloning site (MCS): insertion area = allow DNA to be inserted in vector
• Origin of replication: make multiple copies of self (including the inserted DNA)
• Selectable marker gene (antibiotic resistance): make more copies & outcompete others

Question 6

What to consider when choosing a suitable vector?

Answer 6

properties that will depend on the purpose of cloning and the size of the insert

Question 7

Why are transformed cells allowed to recover in a rich (selective) media?

Answer 7

cells containing the vector will be able to grow (w/ or w/out inserted DNA) bc vectors have selectable marker gene which resists antibiotics

Question 8

How can we use blue-white screening to select our desired fragment?

Answer 8

• MCS is in lacZ gene fragment => metabolise X-gal => produce B-galactosidase=> blue colonies
• but inserted DNA in MCS disrupt gene frag. ≠ metabolise ≠ produce => white colonies

Question 9

What is a DNA library? & name 2 types of libraries

Answer 9

collection of clones that represent a (partial / whole) genome of an organism

• Genomic library
• cDNA library: genes expressed (RNA -> cDNA)

Question 10

Explain how a genomic DNA library prepared?

Answer 10

1. genome cuts into smaller pieces
2. Clone pieces into appropriate vector
3. Insert recombinant vector in bacterial cells
4. Separate colonies w/ diff. genomes

Question 11

How do we generate complete coverage of a whole genome?

Answer 11

1. Partially digest/cut pieces (my limiting no. enzymes or time course)
2. overlap pieces of strands to help sequence pieces of DNA in right order

Question 12

Explain how a cDNA library prepared?

Answer 12

1. Reverse transcriptase mRNA -> cDNA
2. DNA cloning
   * note: highly expressed / active genes will produce more copies

Question 13

What is the purpose of a probe?

Answer 13

Identify a gene during cloning

Question 14

What can be used for a probe?

Answer 14

short ssDNA

Question 15

What’s the difference b/w genetic engineering & DNA cloning?

Answer 15

Engineer: change DNA
Cloning: make multiple identical copies

Question 16

5 steps for cloning

Answer 16

1. obtain desired DNA fragment
2. Prepare suitable vector
3. ligation of fragment (insert) to vector
4. transformation of cells w/ recombinant vector (vector put in cell)
5. Screen the transformed colonies w/ recombinant vector