EXAM 3: DNA Tech

Question 1

Recombinant DNA

Answer 1

DNA from two or more sources is incorporated into a single molecule

Question 2

Restriction enzyme

Answer 2

An enzyme that cuts DNA at or near specific nucleic acid sequences known as restriction sites

Question 3

Plasmid

Answer 3

is an autonomously replicating, circular piece of DNA

Question 4

Vector

Answer 4

A vehicle used to transfer genetic material to a target cell

Question 5

Discuss the applications of Recombinant DNA technology

Answer 5

Definition: Devices, techniques, and procedures that intentionally modify genomes of organisms for specific purposes

Applications:
Can genetically manipulates an organism
Understanding of gene and protein function & pathogenesis
Eliminating of undesirable phenotypes
Creating new organisms or ones that synthesize helpful product (enzymes)

Question 6

Discuss the tools of recombinant DNA technology

Answer 6

1. Restriction Enzymes
   - an enzyme that cuts DNA at or near specific NA sequences known as restriction sites
   - ex: HindIII
2. Plasmid Vectors
   - circular molecule of DNA used to transfer genetic material into a host cell
3. Synthetic NA
   - DNA & RNA created in cell-free solutions
4. Mutagens
   - Physical & chemical agents that cause changes to nucleotide sequences
5. Reverse transcriptase
   - An RNA dependent DNA polymerase

Question 7

Discuss restriction enzymes and their use

Answer 7

“Molecular scissors” that cut DNA at a specific sequence
- used to cut DNA into smaller more manageable sequences

Natural defense against bacteriophage
- “restrict” the invasion of foreign DNA
- Used to digest foreign DNA

Most are palindromic sequences
- Sequences are the reverse complement

Sticky ends
- unpaired portion of DNA that eventually fragments w/ another sticky end

Blunt ends
- non-cohesive, paired end of DNA

Question 8

Characterize artificial plasmids and discuss their use in molecular cloning

Answer 8

Autonomously replicating, circular piece of DNA
– Origin of replication (ori)
– Selectable marker
– Multiple cloning sites

Once cut DNA is inserted into a plasmid it is considered recombinant DNA
- target gene is inserted into the plasmid, which is inserted into bacterium through transformation & more plasmid DNA can be made

Question 9

Discuss natural and artificial methods of inserting DNA into host cells

Answer 9

Natural methods
Transformation: Free uptake DNA by the cell from the environment
Transduction: Transfer of DNA from one cell to another through direct contact
Conjugation: Transfer of DNA from one cell to another via a pilus

Artificial methods (EPI)
* Electroporation: electrical current
* Protoplast fusion: enzymes degrade cell wall
* Injection: gene gun & microinjection

Question 10

Discuss the steps, materials, and applications of PCR

Answer 10

Amplification of DNA in vitro

Steps:
- repeats 30x (2–>4–>8—>12)
1. Denaturation (95 C)
- disruption of H bonds, not covalent bonds
2. Annealing (55 C)
- primer anneals & oligonucleotide binds to template
3. Extension (72 C)
- enzyme is active and nucleotides are added to produce second strand of DNA

Materials needed:
- DNA primers
- dNTPs (deoxy nuc tri phosp)
- Polymerase (Taq thermophile origin)

Applications:
- gene analysis and genetic fingerprinting, etc.

Question 11

Provide an overview of the steps involved molecular cloning

Answer 11

Must find clone containing DNA of interest

Select based on growth conditions
* Resistance genes/ susceptibility genes

Verify gene of interest
* DNA isolation, restriction digest, gel electrophoresis

Confirm
* Sequencing, Western blot analysis, and/or Southern blot

Cloning:
1. Target gene (resistant/susceptibility genes) is selected and inserted into plasmid (DNA ligase)
2. Plasmid is introduced into bacteria via transformation
3. Bacteria containing the plasmid is selected using antibiotics
4. Bacteria with correct plasmid are used to make more plasmid DNA, or induced to express the gene and make a protein