Biotechnology

Question 1

Briefly describe the process of recombinant DNA technology

Answer 1

DNA is used from two different organisms, “cut and paste” methodology

Generally- transferring specifically selected pieces of DNA from one organism to another

• Cuts the DNA with a restriction enzyme (bacteria)
• Each restriction enzyme cuts DNA at a SPECIFIC sequence to form a plasmid vector
• Can be used to make human insulin or hGH
• Inserts genes for each of the two insulin polypeptides next to a highly transcribed gene (e.g. beta-galactosidase)
• Transform the bacteria and express the insulin genes to proteins
• Chemically remove the insulin polypeptides from beta-galactosidase protein
• Combine two subunits to produce insulin

Question 2

Describe the process of obtaining artificial insulin.

Answer 2

• Scientists build the human insulin gene in the laboratory
• Remove a loop of bacterial DNA known as a plasmid
• Insert the human insulin gene into the plasmid
• Return the plasmid to the bacteria
• put the “recombinant” bacteria in large fermentation tanks.
• The recombinant bacteria use the gene to begin producing human insulin
• Harvest the insulin from the bacteria and…
• purify the substance for use as a medicine for people.

High purity

high specific activity with a steady supply

Question 3

Describe the process of PCR.

What is reverse transcription?

Answer 3

Amplifies a specific, known sequence of DNA

Separates the DNA helix and binds the primers to a sequence you want to replicate

Requires primers that match your sequence, dNTPs

DNA pol is used to copy between two primers (taq pol is used in order to not denature)

Denature Anneal Extend

qPCR done in real-time, can use either RNA or DNA

use RT in order to look at RNA

mRNA can be copied into cDNA using RT

RT is a DNA pol that uses a single stranded RNA as a template

PCR uses a fluorescent dye that can be detected

More starting material, faster the amplification of the target RNA/DNA will occur

E.g. the lower the CT value, the higher the expression of the product

Question 4

What is the main principle of gel electrophoresis?

Answer 4

Use of electrical currents to separate fragments into size and charge

DNA is negative and moves towards the positive pole, smaller fragments move faster and farther than larger

Question 5

Distinguish between Northern blot, Southern blot, Western blot

Answer 5

Distinguish between Northern blot, Southern blot, Western blot

Southern Blots

Uses a DNA probe on DNA to identify a sequence of interest

Detect mutation in FG using this blot, short piece of synthetic DNA complementary to the target sequence

Probe with ASO specific for normal CFTR will show up EXCEPT in the homozygous for CF

Northern Blot

Looks at RNA using a DNA probe

Western blot

Uses SDS-Page to separate the proteins, migrates towards the anode (versus the cathode)

Stained gels are on a nitrocellulose sheet that uses specific antibodies added and labeled (antibody probe visualizes the bands)

Question 6

What are the different methods by which gene expression is analyzed?

Answer 6

mRNA analysis

Northern blots, qPCR

Microarray: immobilized DNA sequences, label cDNAs with different fluoresences to determine which spot is saturated with producing this message

Protein analysis

Western Blots, ELISA, Proteomics

Question 7

Describe the process of gene therapy and what are the major risk factors. How can it be useful for ADA disease?

Answer 7

Normal gene is inserted into the genome to replace a disease-causing gene

Can replace a mutated gene, inactivate a gene, or introduce a new gene to fight disease

ADA is essential for lymphocyte development, disease causes a deficiency thus using a retrovirus can integrate this into the DNA so the patient can synthesize the enzyme

Question 8

How are transgenic animals created?

Cut and paste methodology

Question 9

Discuss some pros and cons of biotechnology.

Pros:

Disease resistant crops

New vaccines, bigger livestock

Cons:

Unpredictable, unsure of side effects

Unexpected impacts and concerns for safety

Question 10

The following technology is very important to synthesize artificial insulin

Recombinant DNA technology

PCR

Gel electrophoresis

Gene therapy

Question 11

PCR is used for the following application

DNA cloning

Diagnosis of infectious diseases

Genetic Fingerprinting

Paternity testing

Prenatal diagnosis of disease

All of the Above

Answer 11

All of the above