Genetics Exam 3 - Plasmid Vectors and PCR

Sterile insect technique

Annihilating screwworm population by introducing sterile females (sterilized using ionizing radiation).

Restriction Enzymes

Specific endonucleases, ds breaks at each recognition sequence, used for defense against invading DNA.

Sticky ends

DS break not straight across, overhangs allow for complementary rejoining with original or new strand with complementary overhang. Nicks resealed by DNA Ligase.

HindIII and EcoRI RE

Form sticky end in same point of palindromic sequence, commonly used for vectors.

Uses of Cloning Vectors - Expression Vectors

Produce large amounts of RNA/protein of interest (ex. insulin), includes sequences to allow for expression of genes in heterologous systems (transgenic orgs).

Requirements for a Cloning vector

1. Origin of replication to allow for multiple copies to be produced

2. Selectable markers to identify cells containing vector (usually antibiotic resistance gene)

3. Single cleavage site for each restriciton enzyme used (polylinker/MCS site for versatile DNA pasting).

4. Optional - Lacz marker (splits if foreign DNA is succesfully integrated → white instead of blue colonies)

Steps of Insertion of DNA into vector

1. Digest plasmid with RE (EcoRI)

2. Remove 5’P ends to prevent reannealing to self (phosphatase)

3. Digest froeign DNA with RE

4. Purify

5. Allow fragments to anneal via sticky ends

6. Nicks in backbone sealed by DNA ligase

7. Transform bacteria (uptake of vector)

8. Spread colonies on plate with ampicillin

9. Isolate amp resistant colonies

10. Determine if cloning worked

How to confirm cloning worked

Set up ligation experiment:

Tube 1: EcoRI phosphatase-treated vector (see if cutting worked)

Tube 2: EcoRI cut, phosphatase treated vector + ligase (see if problem with phosphatase)

Tube 3: EcoRI cut, phosphatase-treated vector + purified fragment (negative control)

Tube 4: EcoRI cut, phosphatase treated vector, purified fragment, ligase (experiment)

Restriction Digestion Confirmation of Transformation via Plasmid Vector

Lane 1: uncut vector (slightly further than size marker)

Lane 2: Vector only (cut)

Lane 3: Colony from experiment tube (should have banding at plasmid length and banding at foreign DNA length)

Lane 4: size marker

Uses of Recombinant DNA Tech

Test what genes do (complementation/changes), mass produce medications like insulin

Steps of Polymerase Chain Reaction

1. DNA heated to 100C to separate dsDNA

2. DNA colled to 30C to allow primers to anneal

3. Heated to 70C for polymerase to synthesaize new strands, creating 2 new dsDNA molecules

4. Repeat process for exponential growth

Requirements for PCR

Heat stable DNA polymerase (Taq), thermocycler (rapid temp change), DNA sample, primers matching portion of sequence of interest.

PCR in Plasmid Vectors

Amplification (multiple copies) of foreign DNA promotes likelihood that it will be integrated into the vector, which when added to a transformed bacteria can then be replicated to produce massive amounts of product.

PCR Product and REs

PCR primers also have RE recognition sequence on 5’ ends (not complementary to target sequence), allowing for proper cleavage of produced fragment ends.

Recombinant Tech In Agriculture

Generate transgenic plants that express Bt cry protein toxin to kill insect pests.

Generation of transfenic plants/crops

Target plasmid vector and pathogenic vector transform bacteria → bacteria uses Ti plasmid to infect plant and transfer target plasmid to it → integrates into the plant chromosomes.

Gene from different species/Species with DNA from different source

Transgenic