Module 4: Biotechnology

Question 1

Immunohistochemistry (IHC) is used to:

Answer 1

***IHC relies on antibodies to asses protein spatial distribution as a QUALITATIVE measure… you cannot quantify the amount of protein in IHC but you can determine if it is present

• Demonstrate antigens (most often proteins) in cells and tissues
• in their exact localization
• by using a specific antigen-antibody reaction in which the antibody is labeled by different chemicals
• by preserving the structure of the cells and tissues

Question 2

The principles of IHC ______.

A. use of microscopy and immunology together
B. highly specific binding of antibodies to their own antigens
C. making antibody-antigen complex microscopically visible
D. all of the above

Answer 2

D. all of the above

Question 3

What are the basic steps of RT-PCR and what is it used for?

Answer 3

1. Annealing of downstream primer to RNA
2. Reverse transcription w/ reverse transcripase
3. RNA copied from 3’ to 5’ into cDNA

Question 4

PCR requires the following:

Answer 4

1. Template DNA
   - a sequence of DNA that is to be copied (also called target DNA)
2. Pair of DNA primers
   - primers are short RNA strands that exactly match the beginning and end of the DNA fragment to be amplified
3. DNA polymerase
   - builds a new DNA strand in the 5’ to 3’ direction
   - called Taq polymerase (able to stand heat)
4. dNTPs
   - (deoxynucleosides) are the building blocks
5. Buffer
   - Taq needs Mg++ *******

Question 5

What are the 3 steps in PCR?

Answer 5

1. Denaturation (high temp)
   - breaks hydrogen bonds to separate DNA strand
2. Annealing
   - primer binding to target also called hybridization (low temp)
3. Extension
   - medium temp

Analysis of PCR using Gel Electrophoesis:
- allows you to determine size (smaller molecules move faster)

Question 6

What are 5 uses for PCR?

Answer 6

1. DNA sequencing
2. DNA profiling (fingerprinting)
3. Making recombinant DNA for GMOs
4. Detecting foreign organisms in food salmonella, E. coli
5. Detecting the cause of an infection or disease

Question 7

What is the purpose of immunoblotting?

Answer 7

The purpose of this method is to separate proteins according to their size

Question 8

What are the major differences between each of the following?

• IHC
• RT-PCR
• Immunoblotting

Answer 8

IHC: Qualitative, uses antibodies (proteins)

RT-PCR: Quantitative, used for DNA or RNA (NOT proteins)
- HIGHLY quantifiable

Immunoblotting: Quantitative, uses antibodies, uses gels