DNA manipulation 15

Question 1

endonucleases( restriction enzymes)

Answer 1

Molecular scissors that cut phosphodiester bonds along nucleic acid strands at a specific recognition site.

Question 2

Sticky ends

Answer 2

a staggered cut by a restriction enzyme resulting in overhanging nucleotides

Question 3

blunt ends

Answer 3

a straight cut by a
restriction enzyme resulting in no
overhanging nucleotides

Question 4

Sometimes VCAA exams will ask you to determine how many fragments a piece of DNA will be
cut into given a certain number of recognition sites
- Be careful!

Answer 4

• For circular DNA like plasmids, the
  number of fragments will equal the number of recognition sites.
• For linear DNA, the number of
  fragments will equal the number of recognition sites plus one.

Question 5

Ligases

Answer 5

To join fragments of DNA together by catalysing the formation of phosphodiester bonds between nucleotides to form the sugar-phosphate backbone.

Question 6

Polymerase

Answer 6

an enzyme that
synthesises a polymer from
monomers, forming a
DNA strand from nucleic acids

Question 7

Overview of enzymes scientist uses to manipulate DNA

Answer 7

1. Endonucleoleases( resistriction enzyme)
   -Cut DNA at a specific recognition site
2. Ligases
   - join (or paste) two fragments of DNA
   3.Polymerase
   - Amplify (or multiply) sections of DNA or
   entire polynucleotides

Question 8

DNA–> RNA what is this process

Answer 8

Transcription

Question 9

Purpose of PCR-polymerase chain reaction

Answer 9

• certain section of gene marked by primers is amplified quickly by making multiple identical copies of segments of DNA

Question 10

Taq polymerase

Answer 10

heat resistant polymerase enzyme used for PCR

- binds complementary nucleotides to the single stranded DNA

Question 11

Materials needed for PCR

DND=T

Answer 11

• DNA sample (denatured +amplified)
• Taq polymerase (elongation)
• Nucelotide base ( complementary)
• DNA primers(join)

Question 12

Process of PCR

Answer 12

1. Denaturation: DNA is heated to 94 degrees to break hydrogen bonds+seperate strands=forming single-stranded DNA
2. Annealing: Single-stranded DNA is cooled to 55 degrees and primers bind to complementary sequences on single-stranded DNA
3. Elongation: DNA is heated to 72 degrees and Taq polymerase binds to primer and begins to synthesized new complementary strand of DNA
4. repeat: steps are repeated to crease more copies of DNA

Question 13

How to separate fragments of DNA

Answer 13

1. DNA is prepared using restriction enzyme or PCR
2. DNA samples placed in wells using micropiette, gel made of agrose and standard ladder is loaded in one wel
3. Gell is submerged in buffer
4. Electric current passed through, DNA (-) so will move to (+) electrode
5. Smaller DNA fragments move faster+go further, current switched off, DNA fragments settle into bands based on size
6. Gel is stained with dye to see under UV lamp
7. Bands can be cut from the gel+purified for further use in experiment

Question 14

How to read gels

Answer 14

Use standary ladder: Mixture of DNA fragments with known lengths used as estimate molecular size of other strands.

Question 15

Gel electrophoresis

Answer 15

a technique that separates DNA fragments based on their molecular size

Question 16

2 properties of DNA fragments that allow them to separate

Answer 16

1. molecular size

2. Charge

Question 17

Homozygous on gel electrophoresis

Answer 17

Thicker bands

Question 18

Heterozygous on gel electrophoresis

Answer 18

fragments with 2 different size

Question 19

Recombinant plasmids as vector transform bacterial cells progress

Answer 19

1. treat plasmid w/ restriction enzyme(sticky end)
2. Treat gene of interest w/ same restriction enzyme, sticky ends produced will be complementary to plasmids
3. Mix treated plasmid+ DNA gene, add ligase, now we have recombinant plasmids.
4. (transformation) Bacteria forced to uptake plasmids into cytoplasm by electroporation or heat shock
5. ( Gene cloning) Bacteria multiply by binary fission=> over time there will be bacteria with same gene producing protein of interest

Question 20

The uses of Recombinant plasmid

Answer 20

introduce DNA into organism where it doesn’t naturally occur

Question 21

The function of restriction enzymes in the recombinant plasmid

Answer 21

• used to cut plasmid vector+gene of interest

- allow gene to be inserted into plasmid

Question 22

The function of ligase in the recombinant plasmid

Answer 22

• insert the gene of interest into the plasmid vector

- forms a single piece of DNA

Question 23

Transform bacterial cells ( purpose)

Answer 23

uptake of plasmid by bacterium ( untransformed bacterial-> transformed bacteria-> recombinant plasmid)

Question 24

Commonly used proteins that are made from transformed bacteria include:

Answer 24

• Insulin-mange diabete
  -Erythroprotein-for treatmeant of anaemia
  Growth hormones
  Interferon
  HepatitasB