molecular tool kit

Question 1

in situ hybridization

Answer 1

by using radioactive RNA probes in tissue section followed by autoradiographic detector, we can see where mRNA is expressed in tissue

we visualize the hybridized probe by using radiation

Question 2

immunohistochemistry

Answer 2

shows where protein is expressed in a tissue

literally - antibody tissue binding

tissues are collected and placed on slide, primary antibodies are introduces, secondary antibodies which are binded to florescent tag bind to primary antibodies -> scientists use fluorescent tags to take photos

primary antibodies bind directly to antigen in the tissue

secondary antibodies have florescent tag attached

Question 3

PCR - polymerase chain reaction

Answer 3

• makes copies of a single sequence of DNA
• only looks at one gene - quicker and cheaper
• the four ingredients needed
  
  1. Template DNA
  2. Nucleotides
  3. DNA polymerase
  4. Primers

3 parts of the cycle
1. Denature - high temp. - opens DNA strands
2. Anneal - lower temp - primers bind
3. Elongate - slightly higher temp - polymerase copies DNA

only takes and hour to two to make a trillion coppies

Question 4

gel electrophoresis

Answer 4

helps visualize the DNA that have been separated by size

short pieces of DNA are lower

how to make gel - take carbohydrate powder, mix it with water, heat it, and let it cool

electric current moves DNA through gel

DNA is negatively charged so it is attracted to the positive pole

DNA ladder - known sizes of DNA used to compare other pieces of DNA to

the technique - DNA is loaded into gel, electronic current pulls the DNA through the gel, and the farther the DNA moves, the shorter the DNA sequence

brighter bands = more DNA
higher bands = longer DNA
lower bands = shorter DNA

Question 5

RT-PCR (reverse transcriptase)

Answer 5

RNA is the template

reverse transcriptase enzyme copies RNA to make a DNA molecule

after RNA is copied to DNA it is called cDNA bc it is complementary to the RNA

Question 6

Western Blot

Answer 6

used to separate and ID proteins in order to know what proteins are present within a cell

1. proteins are separated by size/weight using SDS page which is like gel electrophoresis but in polyacrylamide gel
2. primary antibody binds to the protein of interest
3. washing step - washes away any unbound antibodies
4. secondary antibody binds to primary antibody
   
   • secondary antibody are conjugated to enzyme which converts a substrate mixture to produce a chemiluminescent signal that visualizes the protein band