Enzymes used in molecular cloning

Question 1

What is recombinant DNA?

Answer 1

A type of DNA sequence that’s composed of sequences from 2 or more different sources of organisms

Question 2

What are the steps of molecular cloning?

Answer 2

1. cut/copy + paste DNA
2. transfer to host (usually bacteria)
3. selection + replication

Question 3

What is a vector?

Answer 3

A vehicle to carry your gene

Question 4

What are the features common to all vectors?

Answer 4

-origin of replication - independent replication in host
-selectable marker - identify host cells that contain the vector
-multiple cloning site - where to clone the gene

Question 5

What are the 3/4 types of restriction enzymes?

Answer 5

-type I + III - cleave DNA at random, far away from recognition sequence
-type IV - cleave modified DNA
-type II - cut DNA at defined position, either within or near recognition site

Question 6

Describe type II restriction enzymes

Answer 6

-DNA sequence is usually palindromic
-recognise specific DNA sequence
-can generate blunt ends or overhangs
-cleavage generates 5’ phosphate + 3’ OH groups

Question 7

What are overhangs?

Answer 7

Short piece of single stranded DNA - 5’ or 3’

Question 8

How do restriction enzymes work?

Answer 8

-initial binding is non-specific
-enzyme moves along DNA until it finds specific recognition site
-specific binding triggers structural change in DNA + enzyme
-catalysis requires Mg2+
-generates a free 5’ phosphate and 3’ OH

Question 9

What are overhangs compatible with?

Answer 9

-others that have been cut with the same enzyme
-cut vector + insert with the same enzyme

Question 10

What does DNA ligase do and why is it needed?

Answer 10

-no phosphate sugar backbone
-H bonds form between complementary base pairs of sticky ends - temporary interaction
-DNA ligase catalyses formation of new phosphodiester bond

Question 11

What are potential problems of trying to add an insert into the vector?

Answer 11

-vector has complementary ends so might ligase to itself - likely as ends are closer together
-gene may insert in wrong orientation

Question 12

Describe the modification of DNA by adding/removing the 5’ phosphate

Answer 12

-adding a 5’ phosphate may be necessary if there isn’t one e.g PCR
-T4 polynucleotide Kinase is used
-calf intestinal phosphatase (CIP) used to prevent self-ligation of vector

Question 12

What are 2 ways of modifying the ends of DNA?

Answer 12

-addition/removal of the 5’ phosphate
-removing a DNA overhang

Question 13

Describe the modification of DNA by removing a DNA overhang

Answer 13

-fill in 5’ overhang - T4 DNA polymerase or DNA polymerase I
-remove 3’ overhang - T4 DNA polymerase or DNA polymerase I
-remove 5’ overhang - Mung Bean Nuclease

Question 14

What are the steps of transformation?

Answer 14

1. getting plasmid into host
2. selectable marker - usually antibiotic resistance gene

Question 15

What are 2 ways of getting the plasmid into the host?

Answer 15

-electroporation - brief pulse of high-voltage
-chemical transformation - chemically treated E. coli is subjected to heat-shock, causing cell membrane to change so it can take up DNA