L12 Enzymes Used In Molecular Cloning

Question 1

What is molecular cloning?

Answer 1

A set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules

Question 2

What is recombinant DNA?

Answer 2

A type of DNA sequence that is composed of sequences from two or more different sources or organisms, such as synthetic (lab-made) sequences and microorganisms

Question 3

Why is molecular cloning carried out?

Answer 3

1. To isolate a specific region of a DNA we are intrested in - an entire gene, coding sequence of a gene or promoter region

Question 4

What are the steps of molecular cloning? (5)

Answer 4

1. First step is to make recombinant DNA.
2. In molecular cloning, one of the pieces of DNA is a vector (discuss what this is in a moment – basically a carrier).
3. Cut and paste vector to the DNA fragment of interest.
4. Recombinant DNA is not much use unless we can isolate a single species and make more of it. Put it into host – E. coli (“transformation”). Acts like a factory.
5. Selection and host replication – lots of progeny, all containing recombinant DNA.

Question 5

What is a vector?

Answer 5

A vector is a vehicle to carry the desired gene into the host, replicating it and maintaining it over the generations

Question 6

What are common essential features of all vectors?

Answer 6

1. Origin of replication
2. Selectable marker
3. Multiple cloning sites

Question 7

What is the importance of origin of replication?

Answer 7

It is needed for independent replication inside the host

Question 8

What is the importance of the selectable marker?

Answer 8

Survival of host cells that are carrying your plasmid

Question 9

What is the importance of the multiple cloning sites?

Answer 9

It is responsible for where the gene is cloned and restriction enzyme sites.

Question 10

What are the different types of vectors?

Answer 10

1. Plasmid
2. Phage
3. Cosmid
4. BAC - Bacterial artificial chromosome
5. YAC - Yeast artificial chromosome
6. MAC - Mammalian artificial chromosomes.

Need not remember everything

Question 11

What type of vector is most commonly used and why?

Answer 11

Different types of vector exist. Most commonly used ones are plasmids (circular DNA that exists outside of the chromosome in bacteria). Plasmids carry genetic information – often something that is useful for survival!

Question 12

How do you clone the desired DNA into the vector? What is needed?

Answer 12

• Something to cut DNA in a specific place
• Something to stick DNA back together again
• A way to prepare your insert: Cut? Amplify? Purify?
• Modify the DNA ends

Question 13

What is the best tool to cut up DNA?

Answer 13

Restriction enzymes

Question 14

What is the restriction and modification system?

Answer 14

Phage (λ) grown in one bacterial host failed to grow in a different bacterial host: Restricted

Rare progeny λ phage were able to grow in the new host: Modified

Question 15

What is the role of methylation in the restriction-modification system?

Answer 15

Bacteria possess enzymes called restriction enzymes that recognize specific DNA sequences and cut the DNA at those sites.

To protect their own DNA from being cut by these enzymes, bacteria also have methyltransferases.

These enzymes add methyl groups (CH3) to specific bases within the recognition sequences of the restriction enzymes.

This methylation effectively “marks” the bacterial DNA as “self,” preventing the restriction enzymes from cutting it.

Question 16

How does phage DNA escape the restriction-modification system?

Answer 16

Sometimes a rare piece of invading phage DNA acquires the correct methylation pattern - now protected from degradation.
Phage can produce progeny.

Question 17

How many restriction enzymes are known and used?

Answer 17

Thousands are known, but hundreds are currently used.

Question 18

How are restriction enzymes classified and explain them?

Answer 18

Restriction enzymes are broadly classified into three/four types:

1. Type I and III: cleave DNA at random, far away from the recognition sequence.
2. Type IV: cleave modified DNA.
3. Type II: are the useful ones! They cut DNA at a defined position, either within or near to their recognition site.

Question 19

Name the features of type two restriction enzymes

Answer 19

1. Most widely used ones are protein homodimers.
2. DNA sequence is usually palindromic.
3. Recognise specific DNA sequence (usually 4-8bp).
4. Can generate overhangs (5’ or 3’) or blunt ends.
5. Cleavage generates 5’ phosphate and 3’ OH groups.

Question 20

How does a restriction enzyme (BamHI) work?

Answer 20

1. Initial binding is non-specific: looser, catalytic site not involved (no specific cutting)
2. Enzyme then moves along DNA: it can “slide” for short distances but can also jump or hop over longer distances if it doesn’t encounter a specific site
3. Recognition of a specific site - conformational changes (enzyme and DNA). Exact mechanism not yet known
4. Ends have 5’-phosphate and 3’-OH groups

Question 21

What are overhanging ends?

Answer 21

Overhanging ends are “sticky” – can base pair with matching overhanging ends
Any overhanging end generated by EcoRI is compatible with any other.
Can cut vector and insert with same enzyme - compatible overhanging ends

Question 22

Which enzyme is responsible for sticking DNA fragments together?

Answer 22

DNA Ligase

Question 23

What is the role of restriction enzymes in DNA manipulation?

Answer 23

Restriction enzymes cut DNA at specific sequences, allowing for precise manipulation of DNA fragments.

Question 24

What are the types of ends produced by restriction enzymes?

Answer 24

Restriction enzymes can generate two types of ends:

Sticky ends: Short, single-stranded overhangs that can base pair with complementary overhangs from other DNA fragments.
Blunt ends: Double-stranded ends without overhangs.

Question 25

How can DNA fragments with compatible ends be joined together?

Answer 25

Complementary sticky ends can come together and form hydrogen bonds between the complementary base pairs. However, this is a temporary interaction. To form a stable, covalent bond between the DNA fragments, DNA ligase is required.

Question 26

What is the role of DNA ligase?

Answer 26

DNA ligase catalyzes the formation of phosphodiester bonds between the 5' phosphate group of one DNA fragment and the 3' hydroxyl group of another, sealing the gap and creating a continuous DNA molecule.

Question 27

What is the ligation reaction?

Answer 27

Complementary ends interact together and DNA ligase catalyses the formation of a new phosphodiester bond.

Question 28

Explain DNA ligase mechanisms.

Answer 28

1. AMP is transferred to a lysine residue in the enzyme’s active site (from ATP – the cofactor). 2. AMP is then transferred to the 5′-phosphate. 3. The AMP-phosphate bond is attacked by the 3′-OH, forming the covalent bond and releasing AMP. 4. ATP is required to replace the AMP used in the reaction (i.e. is a cofactor)

Question 29

What are the potential molecular cloning issues?

Answer 29

1. There might not be convenient restriction sites. 2. There might have not been enough DNA. 3. The DNA might be mixed in with lots of other DNA molecules.

Question 30

What are the potential problems with the vectors?

Answer 30

1. The vector has complementary ends – it might ligate to itself: * Modify vector ends – phosphatase treatment removes 5’ phosphate – no phosphodiester bond can be formed. 2. Gene may insert in wrong orientation * Use more than one enzyme (also solves first problem) for each end

Question 31

What is the role of phosphate groups in DNA ligation?

Answer 31

5' phosphates are required for ligation. No phosphate = no ligation. * If there are only 5' phosphates on one strand at each end, then only one of the two DNA strands is going to form the phosphodiester bond. * The other strand will have a 'nick' in the DNA but this will be repaired by host enzymes once inside a bacterial cell.  

Question 32

How do we ensure DNA fragments are ready for ligation?

Answer 32

If there is no phosphate, we need to add one for ligation to work. * PCR products usually don't have 5' phosphate groups - ends need to be phosphorylated for ligation to be successful. * Restriction enzyme cut DNA has a phosphate.   * Polynucleotide kinase will catalyse phosphorylation of 5' ends (from ATP).  

Question 33

How can we prevent unwanted ligation?

Answer 33

* Removing a phosphate can prevent unwanted ligation, e.g. self-ligation of a vector during cloning. * Treat cut vector with CIP1 (CIP removes 5' phosphate). Sticky ends can still associate but no covalent bond is formed. Ligation cannot occur.  

Question 34

Why do we remove a DNA overhang?

Answer 34

1.Blunt end cloning might be necessary 2.Destroy restriction enzyme sites

Question 35

What are the several possible strategies for overhang removal?

Answer 35

1. Fill in the 5' overhang - Polymerase. 2. Remove the 5' overhang - exonuclease activity 3. Remove the 3' overhang - Mung Bean Nuclease.

Question 36

What is the last step of molecular cloning?

Answer 36

* Transfer to host * Selection and replication

Question 37

How is transformation done in molecular cloning?

Answer 37

1. Electroporation - brief pulse of high-voltage. 2. Chemical Transformation - Chemically treated e coli, subject to heat shock and causes cell membrane changes that allow uptake of DNA.

Question 38

How do you select bacteria that have been transformed with a plasmid?

Answer 38

* Use the selectable marker on your vector. * Typically, it is an antibiotic resistance gene that allows bacteria to grow on a plate containing that antibiotic. * Each single bacterium will form a colony of identical bacteria containing the plasmid. * Successfully transformed bacteria will contain the vector. As the cells divide, so does the plasmid - hence the name "cloning". 

Question 39

What are the commonly used antibiotics?

Answer 39

1. Ampicilin - Inhibits bacterial cell wall synthesis. 2. Tetracyline 3. Kanamycin