Recombinant DNA and Plasmid Notes

April 19, 2023

Recombinant DNA and Plasmid Notes

Remember DNA?

What is the monomer of DNA?– Nucleotides

How do bases pair? – A – T– C – G

What kind of bond is used?– Hydrogen bonds between nitrogen

bases

I. Restriction Enzymes AKA Restriction

Endonucleases What macromolecule

do you think they are made of?– They are PROTEINS

that cut strands of DNA at specific nucleotide sequences

Restriction Enzymes (cont.)

A. There are many different restriction enzymes that each cut DNA at different nucleotide sequences

B. Most will cut the DNA with a staggered cut

C. Usually occurs at a palindrome: a sequence of units that can be read the same way in either direction

5‘…GAATTC…3’ 3‘…CTTAAG…5’

D. Sticky Ends

1. The staggered cuts leave the DNA with end pieces “sticking off” a. We call these “sticky ends”b. These exposed N-bases will want to

join with other complimentary exposed bases

What If???

What do you predict could happen if two pieces of DNA are cut with the same restriction enzyme???– YES! They will have the same “sticky

ends”

– How could we use this???

E. Types of Restriction Enzymes1. Sticky End- already discussed2. Blunt End

a. These cut the DNA straight across and create blunt ends:

CCC GGGGGG CCC

F. Products generated by restriction enzymes1. COHESIVE END CUTTERS (staggered cuts): Enzyme Recognition Site Ends of DNA After Cut

2. BLUNT END CUTTERS (direct cuts): Enzyme Recognition Site Ends of DNA After Cut

5’…GAATTC…3’3’…CTTAAG…5’

5’…G AATTC…3’3’…CTTAA G…5’

EcoRI

Pst I 5’…CTGCAG…3’3’…GACGTC…5’

5’…CTGCA G…3’3’…G ACGTC…5’

HaeIII 5’…GGCC…3’3’…CCGG…5’

5’…GG CC…3’3’…CC GG…5’

1. Restriction enzymes are named according to the following nomenclature:

Ex: EcoRI E = genus Escherichia

co = species coli R = strain RY13 I = first enzyme

isolated

G. Restriction Enzyme Naming

Why would anyone go through the trouble of cutting DNA???

One reason…– Recombinant DNA

Break down the word…what do you think recombinant means?

Other reasons…– DNA fingerprinting, gene therapy…

II. Recombinant DNA

A. Recombinant DNA: DNA that has been cut from one strand of DNA and then inserted into the gap of another piece of DNA that has been broken.

1. The host DNA is often a bacterial cell such as E coli.

B. Bacterial Structure

1. Bacteria are often used in biotechnology because they have plasmids

2. A plasmid is a circular piece of DNA that exists apart from the chromosome and replicates independently of it.

3. A plasmid is therefore called a VECTOR.

C. Vectors

1. What is a vector?a. Something that is used to transfer genes

into a host cellb. Examples

1. Bacterial plasmids2. Viruses

D. Isolating Genes

1. Must isolate the gene of interest first before you insert it into the plasmid

2. How do you do this?

a. Use a restriction enzyme!!!

E. Final Steps of Making Recombinant DNA1. Once the gene is isolated, have to cut the

organism’s DNA with the same restriction enzyme…why?

a. The sticky ends will naturally be attracted to each other

2. Add DNA LIGASE: enzyme that seals the fragments together

3. Now organism is called a Transgenic Organism- organisms that contain functional recombinant DNA (rDNA) from a different organism

III. Uses for Recombinant DNAA. Recombinant DNA has been gaining importance

over the last few years, and will become more important as genetic diseases become more prevalent and agricultural area is reduced. Below are some of the areas where Recombinant DNA will have an impact:

1. Better Crops (drought & heat resistance) 2. Recombinant Vaccines (i.e. Hepatitis B) 3. Production of clotting factors 4. Production of insulin 5. Production of recombinant pharmaceuticals 6. Plants that produce their own insecticides 7. Germ line and somatic gene therapy

RECAP Steps for making a

transgenic organism:1. Locate and isolate the

gene of interest2. Cut out the gene and

cut the plasmid using the appropriate restriction enzyme

3. Insert the desired gene into the plasmid matching up the sticky ends

4. Use the enzyme DNA ligase to seal up the sticky ends

5. Transfer the vector in the host organism where it will replicate

6. Host organism produces the protein coded for by the recombinant DNA

Insulin Production