Recombinant DNA Technology

Question 1

Discuss the applications of Recombinant DNA technology

Answer 1

Recombinant DNA technology includes Devices, techniques, and procedures that intentionally modify genomes of organisms for specific purposes

Genetically manipulate of an organism:
1. Understanding of gene and protein function
2. Understanding of pathogenesis
3. Eliminating undesirable phenotypes
4. Creating new organisms
5. Creating organisms that synthesize helpful products → enzymes, peptide vaccines, antibiotics etc.

Question 2

Discuss tools of recombinant DNA technology

Answer 2

Restriction enzymes → Enzymes cut DNA at or near specific nucleic acid sequences known as restriction sites

Plasmid vectors → circular DNA molecule used to transfer genetic material to a host cell

Synthetic nucleic acids → DNA and RNA created in cell-free solutions

Mutagens → Physical & Chemical agents that change nucleotide sequences

Reverse Transcriptase → RNA dependent DNA polymerase (creates complementary DNA from RNA template)

Question 3

what are restriction enzymes and their uses

Answer 3

1. Restriction enzymes → enzyme that cuts DNA at a restriction site (specific nucleotide sequence)
2. “Molecular scissors” → cuts DNA into smaller manageable pieces
3. Natural defense against bacteriophages → “restrict’ invasion of foreign DNA and digests foreign DNA
4. They are palindromic sequences → reverse complement (cleaves DNA from two different sources and they are complements of each other)
5. Sticky end → single sided unpaired portion of DNA not bounded but eventually fragment with a complement sticky end from another DNA fragment
6. Blunt end → non-cohesive end of DNA that is paired

Question 4

characterize the two ends of a Restriction enzyme

Answer 4

Sticky end → single sided unpaired portion of DNA not bounded but eventually fragment with a complement sticky end from another DNA fragment
Blunt end → non-cohesive end of DNA that is paired

Question 5

Characterize artificial plasmids and discuss their use in molecular cloning

Answer 5

A plasmid is an autonomously replicating, circular piece of DNA ( origin of replication (ori) selectable marker, and produces multiple cloning sites)
Once cut, DNA is inserted into a plasmid (considered recombinant DNA because there is DNA from two different sources)
- Often used as vectors
-cloning ( explain)

Question 6

explain cloning

Answer 6

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

Question 7

Why are multiple cloning sites important?

Answer 7

its is just a part of the artificial plasmid ( you want lots of options because it needs to be flanking your gene of interest - it gives you multiple options for restriction enzymes that cut once)
Get rid of anything the cuts into you gene of interest, you need something present in the gene of interest and plasmid hopefully without cutting multiple times)
One cut DNA is inserted i

Question 8

why are selectable markers important

Answer 8

always know what you are working with especially when trying to clone. You can do this with a resistance or selectable marker)
Always confirm that the plasmid is where you want it to be

Question 9

what are the natural method of inserting DNA into host cells

Answer 9

• 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

Question 10

What are the artificial methods of inserting DNA

Answer 10

Electroporation: electrical current

Protoplast fusion: enzymes that degrade cell wall

Injection: gene gun and microinjection

Question 11

What are the Steps of PCR?

Answer 11

The Polymerase Chain Reaction (PCR) multiplying DNA in vitro
These are repetitive steps so each time you double the amount of material

• Denaturation → DNA template into single strands, break all the hydrogen bonds in the double helix providing two templates
  (95 C)
• Priming → each original strand given primers for new strand synthesis, Set boundary for defined sequences that will be amplified
  (55 C)
• Extension → extension of new DNA strands from the primers, (72 C)

Question 12

What are the Materials of PCR?

Answer 12

DNA primers
dNTPs → deoxynucleoside triphosphate synthesis (four nucleotides)
Polymerase → often Taq from thermophile (Thermis aquaticus); a modified recombinant form of Taq typically used as a heat stable DNA polymerase.

Question 13

What are the applications of PCR

Answer 13

Molecular cloning
Gene analysis
Diagnosis
Sequencing
RNA and DNA probes
Antisense molecules and gene silencing
Genetic fingerprinting and forensics
Detection and identification

Question 14

Provide an overview of the steps involved in molecular cloning

Answer 14

1. Selecting a clone of recombinant cells
   -Must find clone containing DNA of interest
   - Select based on growth conditions (resistance/susceptibility genes)
   - Verify gene of interest (DNA isolation, restriction digest, gel electrophoresis)
   - Confirm (sequencing, western blot analysis or southern blot analysis)
2. Process
   A.Target gene (resistant/susceptibility genes) is selected and inserted into plasmid (DNA ligase)
   B. Plasmid is introduced into bacteria via transformation

C. Bacteria containing the plasmid is selected using antibiotics

D. Bacteria with correct plasmid are used to make more plasmid DNA, or induced to express the gene and make a protein

Question 15

Why are DNA primers used in PCR?

Answer 15

Because DNA polymerase can add a nucleotide only onto a preexisting 3’-OH group, it needs a primer to which it can add the first nucleotide.