DNA Manipulation

Question 1

Endonuclease

Answer 1

An enzyme that occurs naturally in bacteria and can cut DNA at a particular recognition site.

Question 2

Ligase

Answer 2

An enzyme that joins together two molecules or fragments of.

Question 3

Polymerase

Answer 3

Enzymes that catalyse the formation of polymers. In nucleotides, polymerase polymers are formed with the use of this enzyme with the use of complementary base pairing rules and with a template strand.

Question 4

Polymerase chain reaction

Answer 4

A technique used to amplify pieces of DNA very quickly.

Question 5

Steps in PCR

Answer 5

1. Denaturation: sample is heated to 90°C + to break the hydrogen bonds between the two strands of DNA to make it single stranded.
2. Annealing: temperature is cooled to 50-60°C to allow for primers to bind to complementary sequences on opposite strands of each target DNA sequence.
3. Extension: temperature is increased to 72°C to allow for Tap polymerase to attach to the primers and then mole along each strand adding free nucleotides to form double stranded DNA.

Question 6

Gel electrophoresis

Answer 6

Allows for samples of DNA to be sorted by size. This information can be useful in DNA profiling, or to isolate a particular fragment for use in DNA recombinant and bacterial transformation.

Question 7

Steps in gel electrophoresis

Answer 7

1. Preparation of gel.
2. Gel placed into a gel electrophoresis chamber with wells at the negative end.
3. Sample is loaded.
4. DNA ladder is used.
5. Gel is placed in bath and covered in ph solution that contains ions.
6. Power sports is attached.
7. Smaller fragments move faster through the gel, so they migrate further through the gel than larger pieces.
8. Stain is used so DNA can be seen.

Question 8

Sticky end restriction enzymes

Answer 8

Leaves fragments with overhanging ends that have exposed bases.

Question 9

Blunt end restriction enzymes

Answer 9

Enzymes that cut DNA to leave flat end fragments

Question 10

Plasmids

Answer 10

Are small and circular pieces of double stranded DNA found in bacterial cells. They replicate independently of the bacterias chromosomal DNA.

Question 11

Recombinant plasmids

Answer 11

Plasmids that have had DNA inserted into them. The sticky end or blunt end enzyme is used to cut both the targeted gene and the plasmid, and then DNA ligase is used to join them together.

Question 12

The importance of antibacterial resistance of recombinant plasmids

Answer 12

Only bacterial cells with the recomibnat plasmids with survive if placed on antibacterial agar plate. This helps ensure that the recombinant plasmids are identified.

Question 13

Steps in bacterial transformation

Answer 13

1. Gene uptake
2. Selection of transformed bacteria
3. Identification of transformed colonies

Question 14

Creating recombinant DNA

Answer 14

1. Cut DNA using sticky end restriction enzyme and then isolate it.
2. Cut bacterial plasmid with same enzyme to make the same sticky ends that are complementary if exposed to each other.
3. Place DNA and plasmids are placed together. (Some plasmids will close up without the DNA).
4. DNA ligase is added to rejoin the sugar-phosphate backbone of DNA.