Week 4 - manipulation of DNA, Nucleases

Question 1

What are Nucleases?

Answer 1

They are enzymes that cut DNA, Exonucleases cut phosphodiester bonds and remove nucleotides at the 5 or 3 end, while endonucleases cut phosphodiester bonds within DNA strands

Question 2

Give examples of Exonucleases and Endonucleases

Answer 2

Exo - Bal 31, removes nucleotides from both strands. Exonuclease 3 removes only from the 3 terminus
Endo - DNasel cuts ss and ds DNA, S1 nuclease cuts only ss DNA

Question 3

What are RE’s and what creation are they responsible for?

Answer 3

RE’s are restriction endonucleases, which allow for the creation of recombinant DNA molecules.

Question 4

Explain RE’s?

Answer 4

RE’s are a type of bacterial immune system used to protect against phage infection, they bind to phage DNA and then cut it causing it to become inactive

Question 5

RE type 2 is the main class of RE used, list some of its uses:

Answer 5

1. Cutting genomic DNA or cDNA to make sticky ends
2. Creation of DNA fragments that can then be ligated into a vector
3. allows for manipulation
4. Study of Gene and protein function

Question 6

List some specific qualities of RE 2 cuts

Answer 6

Site specific cuts
recognition sites are 4 -6 nucleotides long
RE creates breaks in both DNA strands, creating 5’P and 3’OH termini
can produce either blunt or sticky ends

Question 7

How can RE’s be blocked and why?

Answer 7

RE’s are blocked with DNA methylation a process of adding CH3/Me to a base preventing cutting, this is done to protect the organisms own DNA

Question 8

Methylases

Answer 8

Specific and general

Question 9

how many times does a RE cut a DNA molecule

Answer 9

A re that recognise a 4bp long sequence will cut on average every 256, for a 6bp one every 4096bp

Question 10

How is DNA separated in an agarose gel electrophoresis

Answer 10

by size and conformation