Recombinant DNA Technology

Question 1

Production of insulin

Answer 1

1. Extract and purify mRNA from human pancreatic beta cells
2. Convert mRNA to cDNA
3. Amplify the insulin cDNA by PCR
4. Cut insulin cDNA w/ restriction enzymes
5. Isolate plasmid DNA from bacterium
6. Cut plasmid w/ restriction enzymes
7. Ligate insulin cDNA and plasmid
8. Introduce recombinant plasmid DNA into bacteria and grow large amounts of human insulin-expressing bacteria
9. Extract and purify recombinant human insulin from bacteria

Question 2

PCR

Answer 2

starts w/ a mixture of DNA molecules and produces many copies of one specific DNA sequence

denaturing, annealing, and extension

Question 3

Denaturing

Answer 3

breaking of DNA through heating (94-95 deg. Celsius)

Question 4

Annealing

Answer 4

Cool (37-65 deg. Celsius) and anneal primers to complementary sequences w/ their 3/ ends facing each other, flanking target DNA

Question 6

What are the three DNA sources?

Answer 6

genomic DNA, cDNA, chemically synthesized oligonucleotides

Question 7

gDNA

Answer 7

includes introns

Question 8

cDNA

Answer 8

complementary DNA derived by action of reverse transcriptase from mRNA template

Question 9

Restriction enzymes

Answer 9

digest DNA; are of bacterial origin and are involved in defense

Question 10

Probability of occurrence of restriction sites for restriction enzymes in DNA w/ randomly distributed nucleotide pairs

Answer 10

(1/4)^n

Question 11

What do restriction enzymes produce?

Answer 11

They cut DNA at specific sites and some produce blunt ends, overhanging (5 or 3) sticky ends

Question 12

What seals the gap after restriction enzymes cut DNA?

Answer 12

DNA ligase

Question 13

What is the new DNA called after DNA 1 and DNA 2 are joined together?

Answer 13

recombinant DNA

Question 14

What is the DNA cloning process?

Answer 14

Insertion into vector, complementary restriction ends joined by DNA ligase

Question 15

Insertion into vector

Answer 15

cloning into a vector could be accomplished multiple different ways that do not require restriction digestion; gateway cloning, golden gate cloning

Question 16

Plasmid vectors

Answer 16

small circular DNA of bacterial origin, distinct from main chromosomal DNA

have origin of replication; replicate independently of bacterial chromosome

have several engineered characteristics

Question 17

Cloning vector

Answer 17

high copy number per cell; variable insert length; some plasmid have multiple cloning site (polylinker); drug resistance marker-assist in selection; lacZ gene (visual selection for the presence of an insert)

Question 18

lacZ gene

Answer 18

encodes beta-galactosidase; visual selection for vector insert

Question 19

Beta-galactosidase

Answer 19

converts a colorless substrate (X-Gal) to blue color

Question 20

What is the color of the colony with the insert and without?

Answer 20

With: white; lacZ not functional

Without: blue; lacZ converts X-Gal to blue die w/ no insert