DNA Manipulation

Question 1

Gel Electrophoresis Process

Answer 1

1. DNA of interest purified from cells and amplified by PCR to create large sample
2. DNA exposed to restriction enzymes to cut DNA into fragments
3. DNA loaded into wells at negative end of gel and power supply is turned on
4. Short fragments travel further, fragments travel towards positive electrode as DNA is negatively charged (voltage and concentration of agarose gel can also affect how far fragments move through gel)
5. DNA ladder can also be added to gel to compare unknown fragments to fragments of known lengths

Question 2

PCR

Answer 2

1. Denaturation occurs at 95 to break hydrogen bonds and separate DNA strands
2. Annealing occurs at 55 to allow primers to join to DNA strands
3. Extension occurs at 72 which is the optimal temperature for Taq polymerase to add complementary nucleotides to the DNA strands and create double stranded DNA

Question 3

Recombinant Plasmid Process

Answer 3

1. Restriction endonuclease used to cut target DNA and plasmid to create complementary stick ends
2. Target DNA and plasmid combined with ligase to join the DNA into the plasmid
3. Plasmids are inserted into bacteria via heat shock or electroporation which both serve to create pores in the bacterial membrane through which plasmid can enter
4. A selectable marker, such as antibiotic resistance or fluorescence, is used to determine whether the bacteria have become successfully recombinant

Question 4

Insulin Production

Answer 4

1. Genes for Insulin A and B chains are isolated from each other
2. Restriction endonuclease used to cut insulin genes and plasmids to create complementary stick ends
3. Insulin A chain gene is inserted into one plasmid, insulin B chain gene is inserted into a different plasmid using ligase to join the DNA into the plasmid.
4. Each plasmid is inserted into different bacteria via heat shock or electroporation which both serve to create pores in the bacterial membrane through which plasmid can enter
5. A selectable marker, such as antibiotic resistance or inserting insulin gene next to beta-galactosidase protein, is used to determine whether the bacteria have become successfully recombinant
6. Insulin A and B proteins are purified and joined to create functional insulin