Ch 21 - Recombinant Technologies

gel electrophoresis

• a way to visualize and detect (amplified) products

• fluorescent dyes bind to double stranded DNA

• used to separate molecules based on size

Is DNA negatively or positively changed?

negatively

How does gel electrophoresis work?

• nucleic acids are loaded into wells in a gel matrix that is in a salt buffer

• an electrical charge is passed through the gel

• nucleic acids will travel towards the positive pole

• something is added to the DNA or gel that allows for visualization (i.e. EtBr radioactive label)

How can you determine the size of a DNA with gel electrophoresis?

• by running the DNA molecules alongside a “ladder” (a known standard size)

• smaller fragments will travel faster than longer ones

restriction enzymes (restriction endonuclease)

• isolated from bacteria (use restriction enzymes for viral defense)

• cut the dsDNA in a sequence specific way at a recognition site

recognition site

• a sequence 4-8 bp long where a restriction enzyme cuts dsDNA

• a palindromic sequence

palindromic sequence

a sequence that reads the same in 5’ to 3’ direction on both strands (ex: AAGCTT)

Where do restriction enzyme names come from?

the bacteria that they were isolated from

What kinds of cuts can restriction enzymes make?

• staggered or “sticky ends”

• blunt ends

What can restriction enzymes be used to make? How?

• recombinant DNA

• cutting different sources of DNA with the same “sticky end” restriction enzyme allows them to be recombined

When was gel electrophoresis created?

1937

When were restriction enzymes discovered?

1960s

When was recombinant cloning created?

1972

When was Sanger sequencing created?

1975; 1977

When was PCR created?

1983

vector

an agent that can carry DNA into a cell or organism

Where are vectors derived from

from plasmids, bacteriophages, or viruses

How is a target DNA sequence amplified with recombinant plasmids?

• component cells are able to get extracellular DNA from the environment, usually by having a permeable membrane

• can make lots of the sequence with cloning vectors

plasmid cloning

• both the DNA you want to clone, and the plasmid are cut with the same “sticky end” restriction enzyme

• mix the cute sample DNA and the cut plasmid together with DNA ligase

• once the target sequence is in the plasmid, the plasmid is introduced to bacteria through transformation

What are the 4 essential components of a plasmid vector?

1. MCS (multiple cloning site)

2. ori (origin)

3. selectable marker (amp^R)

4. screenable marker (Lac⁺)

multiple cloning site (MCS)

• where the target DNA sequence goes (poly linker)

selectable marker

• uses amp^R gene to identify if the plasmid has successfully been taken up by bacteria cells

• amp^R provides resistance to ampicillin

• only cells that have taken up the plasmids with this gene will survive in the presence of ampicillin

screenable marker

• uses LacZ+ (codes for B-galactosidase and contains the MCS)

• shows whether the target DNA has recombined with plasmid

  • insertion of the target DNA disrupts Lac+ and B-galactosidase becomes disfunctional

How can you detect if the Lac+ gene has been disrupted?

• with X-gal (which is broken down by B-galactosidase)

• if the Lac+ gene has not been disrupted by the target DNA sequence:

  • B-galactosidase is produced a break down X-gal

  • cells appear blue when X-gal is cleaved (THE CELLS YOU DONT WANT)

• if the Lac+ gene has been disrupted by the target DNA sequence:

  • B-galactosidase is not produced, and X-gal is not broken down

  • cells appear white because X-gal has not been cleaved (THE CELLS YOU WANT)

expression vectors

have all the components of a standard vector plus sequences for transcription and translation

Can vectors be used in eukaryotic cells? If so, how?

• yes

• need the appropriate eukaryotic regulatory sequences