• What characteristics make plasmids easy to genetically modify?

  • Plasmids have short sequences of DNA

  • Plasmids are easy to cut and paste back together

  • Plasmids copy independently

• Explain the process and all reagents used to extract DNA.

  1. Lysozyme is added degrade peptidoglycan in cell walls

     • We don’t use lysozyme because we have animal cells

  2. Detergents are added to lyse (explode) the cell

     • Detergents include sarkosyl or sodium dodecyl sulfate (SDS)

     • Proteases and salts are added to clump debris

  3. The mix is then centrifuged to create a precipitate and a supernate

     1. The precipitate is at the bottom and is debris

     2. The supernate is at the top and has the DNA

     3. DNA can be extracted with alcohol

        

• Explain how PCR and Gel Electrophoresis are used in genetic engineering.

  • PCR- Stands for Polymerase Chain Reaction, used to copy DNA many times

    • PCR uses primers which target specific sequences in DNA

    • DNA strands are split apart at 95°

    • Then primers bind to the split strands at 50°

    • The polymerase enzyme makes a complementary strand using free nucleotides at 72°

      

• Gel Electrophoresis- Separates DNA fragments based on size

  • There is a gel made of agarose that has wells (holes) in it

  • DNA is placed into the wells

  • An electric charge is run through the gel

  • DNA is negatively charged and it will move to the positively charged side

  • Smaller fragments will move faster so they will go further

Vocabulary:

genetic engineering- manipulation of genetic information

multiple cloning sites- series of unique restriction enzyme recognition sites

vectors- Vessel to carry genetic information with (often a plasmid)

lysosome- degrades peptidoglycan in cell walls

precipitant- molecular debris, found at bottom of test tube

supernatant- suspends DNA, found at top of test tube

lysed- to explode cells

detergent- Solutions to lyse cells with

protease- Degrades proteins

salts- clumps cellular debris

probes- single-strands of DNA or RNA that bind to a complementary sequence that have a biological marker attached. Shows location of DNA sequences.

hybridization- Process where probe binds to complementary sequence

restriction enzymes- cuts DNA at specific sites

gel electrophoresis- separates DNA fragments based on size

DNA polymerase- Synthesizes new DNA strands in the PCR process

thermal cycler- Used in PCR to take DNA through temperatures best for replication (95°, 50°, 72°)

polymerase chain reaction- used to replicate specific sequence of DNA many times

• We do not replicate all of the DNA

southern blotting- transfers DNA from gel onto membrane with probes. Uses photographic film to identify specific sequences of DNA

exons- coding sequences that leave the nucleus

introns- non-coding sequences of DNA that cannot leave the nucleus