Schwabe Enzymes that manipulate DNA Gene Cloning DNA replication part 1

Question 1

Enzymes:

Answer 1

• Proteins that catalyse biochemical reactions

Question 2

Enzymes that manipulate DNA

x6

Answer 2

• Nucleases
• Ligases
• Polymerases
• Kinases & phosphatases
• Methylases & demethylases
• Recominases / topoisomerases

Question 3

Enzymes that cleave DNA:

Answer 3

Exonucleaes

Endonucleases

Question 4

Exonucleaes:

Answer 4

nibble ends / degrade DNA from 1 end or other

Question 5

Endonucleases:

Answer 5

cut DS internally
• Some sequence- specific => blunt ends across both strands, some staggered cuts => sticky ends
• Ligate => join together

Question 6

Restriction Endonucleases:

Answer 6

protective role in microorganisms => recognise inverted repeats ‘palindromes’ because they bind to DNA as dimers normally cut up foreign DNA.

Question 7

Example of Restriction Endonucleases:

Answer 7

E.g EcoRI from E.coli, BamHI from bacillus amyloliquefaciens, HindIII from Haemophilus influenza etc

Question 8

palindromes are important to

Answer 8

render DNA immune to own restriction enzymes. => does not contain AATT

Question 9

DNA fragments with same sticky ends can

Answer 9

• generate recombinant DNA & basis of gene cloning

Question 10

Cloning process

Answer 10

= isolate DNA from 1 organism & propagate it in another.
⇨ transplanting DNA restriction fragments
⇨ Restriction fragments are produced from donor DNA & vector is linearized using same endonuclease
⇨ LIGATED => polulation of circular RECOMBINANT PLASMIDS
⇨ Introduces to host cells by TRANSFORMATION
⇨ Growth of cells ‘ TRANSFORMANTS’
⇨ SELECTION / SCREENING

Question 11

CLONE =

Answer 11

organism containing foreign DNA by SELECTION or SCREENING can be identified.

Question 12

CLONING VECTOR =

Answer 12

more DNA between cells, circular PLASMID most common as stable in chosen recipient strain.

Question 13

Polymerase Chain reaction – PCR

Answer 13

replicates DNA fragments

Question 14

Melselson & Stahl 1958 DENSITY GRADIENT CENTRIFUGATION

Answer 14

=> Grow E.coli in heavy medium & at time zero shift to light medium (14 NH4Cl)
=> prepare DNA after 0,1,2 generations & centrifuge
=>One generation after density shift i.e. 1 replication cycle : single band of DNA of intermediate density
=> After 2 generations = two bands of DNA 1 light & 1 intermediate density

=> conclusion DNA replication is semi-conservative