Enzyme + Restriction Mapping

Question 1

Describe what nucleases are

Answer 1

nucleases: an enzyme which cleave phophodiester bonds between nucleotides
- ribonuclease
- DNA nuclease

Question 2

Describe what restriction enzymes are, where they are derived from, their natural function and how this is used in molecular biology

Answer 2

definition: catalyse the hydrolysis of phosphodiester bonds between specific, recognisable sequences
naturally produced in bacteria and cleaves infectious viral DNA
function: recognise and cleave specific sequence. Namd based off bacteria of origin
application: used to cleave specific sequences EcoR1 - 5’…GAATTC…3’
palindromic: same sequence both ways

Question 3

Describe applications for restriction enzymes in molecular diagnostics

Answer 3

• cloning
• molecular diagnostics
  (eg: detecting sickle cell anaemia)
• plasmid characteristics
• 5’ or 3’ overhangs form = sticky ends
• blunt ends = cleaved straight down middle (both sequences)
• allows joining of DNA molecules from different sources
• POSITION 6 change from Glu -> Val = deletion of DDE1

Question 4

Describe restriction mapping in detail

Answer 4

• mapping restriction sites w/in a molecule
• utilised in describing plasmids
• separate plasmid samples using separate restriction enzymes (separate digests)
• incubate 1hr 37 degrees
• gel electrophoresis including (standard size lane) example lane for comparison - which determines size of obtained fragments

Question 5

Describe other enzymes used when creating recombinant DNA:
DNA ligase
DNA polymerase
Phosphatases
Polynucleotide kinase
Reverse transcriptase

Answer 5

-DNA ligase: enzyme which catalyses phosphodiester bonds - used to join DNA fragments cleaved by restriction enzymes

-DNA polymerase: joins nucleotides together = useful for blunt or sticky ends caused by even/uneven cleaving

-Phosphatases: catalyse breaking of phosphate bonds????

-Polynucleotide kinase:

-Reverse transcriptase: used to implement foreign viral DNA from vendor into expressing via host cell

Question 6

Give example of recombinant proteins and transgenic organisms used within genetic engineering

Answer 6

Recombinant proteins:
- insulin
- interferon : therapy decapcitates bone marrow therefore ⬆️ stem cell
- G-CSF

transgenic organisms:
- mice
- plants