Lecture 3 - Restriction Enzymes, Ligation, DNA Transfer

Question 1

What are restriction enzymes and what are they used for in bacteria?

Answer 1

1. Recognize specific sequences of nucleotides in a DNA and
2. Cut (by hydrolysis of phosphodiester bond) the DNA into fragments at these sites or more randomly

• Bacteria uses restriction endonucleases to protect themselves by cleaving viral genomes during viral attacks
• Bacteria can protect themselves by modifying their own DNA via methylation (CHO of specific adenine or cytosine bases using methylase enzymes

Question 2

How do restriction enzymes work?

Answer 2

• Restriction enzymes recognize and bind to specific sequences of DNA, called restriction sites
• When it finds its target sequence, a restriction enzyme will make a double-stranded cut in the DNA molecule in specific ways

Question 3

What are the factors affecting RE activity?

Answer 3

Temperature
Amount of enzyme
Duration of enzyme activity
Cofactors
Ionic conditions
Buffer composition
Methylation of DNA

Star activity: Alteration in the digestion specificity that occurs under sub-optimal enzyme conditions.
Results in cleavage of DNA at non-specific sites

Question 4

What is the benefit of using multiple restriction enzymes?

Answer 4

Direction of insert will not be reversed

Question 5

What to consider in the selection of restriction enzymes?

Answer 5

Double digest plasmid and insert with different restriction enzymes at each ends
• Co-digestion
• Sequential digestion

Restriction enzymes have very specific digestion conditions
Reaction buffer and digestion conditions are optimal for both enzymes

NEBcloner website helps you pick restriction enzymes that work well together

Question 6

What kind of ends are involved in ligation?

Answer 6

1. Sticky ends: Same complementary tails in insert and vector DNA
2. Blunt ends: all the complementary nucleotides are base paired

DNA ligase is a ligating enzyme that can join any two cohesive or blunt DNA ends by forming phosphodiester bonds between adjacent nucleotides

Question 7

What are the features of ligation?

Answer 7

Requires Mg2+ and ATP
DNA ligation takes place in 3 steps
DNA ligase does not join single stranded DNA
Blunt end ligation require higher DNA concentration than sticky end ligation

Question 8

What are the 2 possible outcomes of ligation?

Answer 8

Formation of recombinant plasmid
Plasmid self-ligation regenerating original plasmid

Question 9

How to enhance ligation efficiency?

Answer 9

Dephosphorylation of vector
• Prevent reformation of phosphodiester bonds between 5’ and 3’ end of digested vector

Use 2 restriction enzymes to produce non-complementary ends in the plasmid and insert

Insert to Vector Ratio
• The number of insert molecules relative to the number of vector molecules in the reaction mixture

Different ratios ranging from 1:1 to 15:1
• 3:1 ratio is a good starting point for
sticky end ligation
• 10:1 ratio is a good starting point for
blunt end ligation

Optimal ratio determined experimentally

Question 10

How does recombinant DNA transfer occur?

Answer 10

• Delivery of the foreign DNA into the host cell
• Nucleic acid pass through cell membrane pass through cytoplasmic compartment, reach the interior of the nucleus
• Nucleic acid needs to be protected from degradation

Question 11

What is transformation and transfection?

Answer 11

Transformation - introduction of foreign DNA into bacterial cells
Transfection - introduction of foreign DNA into mammalian cells

Question 12

When recombinant DNA enters bacteria cells, it may be:

Answer 12

Degraded by nucleases
Integrated into the chromosome
Co-exist as a plasmid with chromosomal DNA

Question 13

What are the evolutionary advantages of transformation for bacteria?

Answer 13

• Enable bacterial populations to overcome great fluctuations in population dynamics
• Survive harsh and extreme environmental changes
• During such conditions, some bacterial genera spontaneously release DNA from the cells into the environment
• Taken up by the competent cells
• Bacterial cells can be competent by chemicals or electrical pulses

Question 14

What are the steps for bacterial transformation?

Answer 14

1. Bacterial cells are converted into competent cells through treatment with calcium chloride
2. Bacterial cells and DNA are mixed and incubated on ice for 5—45 mins
3. A heat shock for 25-45 s at 37-42 C is applied onto the bacteria cells
4. Incubation on ice for 2—5 min
5. heat shocked bacterial cells are allowed to recover in antibiotic free media for screening and selection

Question 15

What are the factors affecting transformation efficiency?

Answer 15

1. Types of Recombinant DNA
   • DNA exist in numerous conformations
   • Supercoiled DNA is most efficient for transformation
   • Linear or single stranded DNA has very low transformation efficiency
2. Size of DNA
   • The larger the plasmid, the lower the transformation efficiency
3. Methods of recombinant DNA transfer
   Physical methods
   Chemical methods
   Biological methods
   Viral vector-based gene transfer

Question 16

What are the features of physical methods of gene transfer?

Answer 16

• Widely used in prokaryotic and eukaryotic cells
• Direct transfer of DNA into the cytoplasm or nucleus of the host cell
• Safer and consumes less time and labor
• Expensive as it requires specialized instruments which create a physical force that delivers the DNA into the host cell

Question 17

Physical methods of gene transfer
How is biolistic gene transfer (gene gun) used?

Answer 17

• Nucleic acid is coated with high density carrier particles like gold, platinum, tungsten, forming microspheres
• Using high pressure inert gases or high voltage electric discharge, these microspheres are bombarded onto the target cell nuclei

Question 18

Physical methods of gene transfer
How is electroporation used?

Answer 18

• Pores form on the cell surface when exposed to strong electric field
• Pores are transient in nature and reseal without causing damage to the cell membrane structure or affecting the viability of the cell
• Efficiency of nucleic acid transfers include electroporation conditions, host cell and nucleic acid concentration

Question 19

What are the features of chemical methods of gene transfer?

Answer 19

• Cationic polymers, cationic lipids, calcium phosphate, etc. are used in chemical transfection
• The positively charged chemicals form a complex with negatively-charged nucleic acids
• Complexes are attracted toward the negatively charged cell membrane and pass through it

Question 20

Chemical methods of gene transfer
How are liposomes used?

Answer 20

• Liposomes are bilayered synthetic membrane structures composed of lipids
• Nucleic acid and lipids are mixed in a certain ratio
• Liposomes encapsulate the nucleic acid interact with the host cell and enter it
• Liposomes provide a stable environment to the nucleic acids and protect it from enzymatic degradation

Question 21

What are the biological methods of gene transfer?

Answer 21

• High efficiency and specificity
Protoplast fusion
Agrobacterium-mediated
Viral vector-mediated transduction

Question 22

What are the features of viral vector-based gene transfer?

Answer 22

• Viruses are used as gene carriers — also known as transduction
• High rate of replication and protein expression
• Virus particle in which the gene of interest is packaged, enters host cell through a receptor-mediated process
• Applications in gene therapy
• Common viral vectors used for gene delivery include adenoviruses and lentiviruses