BSCI223: Cloning

Why do we use bacteria for cloning?

• Bacteria grow very quickly

• E. coli genome is relatively well understood

• Small plasmids replicate quickly

What are Plasmid Vectors?

Delivery vehicle for gene of interest

What is the structure of Plasmid Vectors?

• Small, circular nucleic acid sequence

• Contains origin of replication

• Contain recognizable marker to allow for identification of bacterial cells that carry the vector (ie. antibiotic resistance gene, metabolism gene)

What are Restriction Enzymes?

Cut DNA molecules in a “restricted” way at specific sequences called “restriction sites” (usually palindromic sequences (same when read forward or backward)); usually named by the bacteria in which they were discovered

What produces Restriction Enzymes?

Bacteria

What are the 2 types of Restriction Enzymes?

Sticky and Blunt end

What type of restriction enzyme does this image show?

Sticky End

What type of restriction enzyme does this image show?

Blunt End

What is a Plasmid Map?

A diagram that represents the genetic structure of a plasmid, showing restriction sites, gene loci, and the origin of replication.

In a plasmid map, what do "Restriction Sites" indicate?

Specific locations where restriction enzymes cut the DNA. A map may show 1 or more of the same restriction site per plasmid.

What does "MCS" stand for on a plasmid map?

They are Multiple Cloning Site; it is a region containing many unique restriction sites used for inserting foreign DNA

What are the three Gene Loci typically highlighted on a standard cloning plasmid map?

bla, lacZa, ori

What is the fundamental purpose of the Polymerase Chain Reaction (PCR)?

It is a method to rapidly amplify a specific DNA segment (gene of interest), creating many copies from very few

What are two primary uses of PCR mentioned in the slide?

• To generate enough DNA for cloning

• To identify whether a specific DNA sequence is present in a genome

How can PCR assist in the cloning process regarding restriction sites?

PCR can be used to introduce restriction sites at the ends of amplified fragments, which allows them to be inserted into plasmids.

Polymerase Chain Reaction requires what 4 things?

1. DNA template

2. Pair of synthetic primers

3. dNTPs (dATP, dCTP, dTTP & dGTP)

4. Thermostable DNA Polymerase

What are the two main methods of in vitro bacterial transformation?

Chemical Transformation and Electroporation

Describe the process of Chemical Transformation

1. Incubate chemically competent cells with DNA (plasmids)

2. Heat shock the cells (ie. 42°C, 30 sec)

3. Allow for recovery

4. Perform positive selection.

Why can't you directly clone genomic DNA from a eukaryote into a prokaryote to produce a protein?

Eukaryotic genes contain introns (non-coding regions). Prokaryotes lack the machinery (spliceosomes) to process pre-mRNA and remove introns

What is the solution to cloning eukaryotic genes into bacteria?

Use Reverse Transcriptase to convert mature mRNA (which has already had its introns spliced out) into cDNA (complementary DNA)

What is the function of Reverse Transcriptase?

It is an enzyme that synthesizes a DNA strand (cDNA) using an mRNA template

What is Positive Selection?

A method that kills all cells except those with the desired phenotype

What is Negative Selection?

A method where only those cells expressing the desired phenotype are killed

What is a Screen?

A method that allows you to pick between different organisms based on different cell phenotypes expressed