UNIT 3 - AOS1 - CH 2.2 & 2.3 - Use of enzymes to manipulate DNA

Question 1

6 tools used by genetic engineers

Answer 1

Synthesise DNA (Polymerase, reverse transcriptase)
Join DNA (ligase)
Cut DNA (Endonuclease)
Edit DNA (CRISPR-Cas9)
Amplify DNA (PCR)
Seperate DNA (Gel electrophoresis)

Question 2

Summarise the 3 types of polymerase

Answer 2

RNA POLYMERASE:
- Runs along DNA, creating a complementary RNA strand. (A,U,C,G)
- Primarily used in transcription

DNA POLYMERASE:
- Runs along DNA, creating a complementary DNA strand (A,T,C,G)
- Used in the amplication of DNA

REVERSE TRANSCRIPTASE:
- Runs along mRNA creating a complimentary copy DNA strand (cRNA)

Question 3

Describe the steps of reverse transcriptase

Answer 3

STEP ONE:
mRNA isolated from cytosol of specific cells

STEP TWO:
Poly-A-Tail added to this mRNA 3’ (provides anchor to primer)

STEP THREE:
Oligo-dT primer added & bind to Poly-A-Tail. Reverse transcriptase enzyme added & DNA lengthens by addition of nucleotides. (order control by mRNA sequencing)

STEP FOUR:
When DNA chain is complete, mRNA is removed by alkali treatment.

STEP FIVE:
Polymerase enzyme added = catalyses the building of complementary DNA strand.

STEP SIX:
Final double-stranded DNA product.

Question 4

Describe Endonucleases

Answer 4

CUT DNA

Restriction enzymes:
- Cut at specific sites based on the nucleotide sequences.
CRIPSR:
- Bacterial defence system/gene editing tool.
- Cut at specific sites based on location of the PAM sequence.

Question 5

Types of ends cut from endonuclease

Answer 5

BLUNT ENDS:
Produced when two DNA strands are cut with no overhang on one or the other strand.

STICKY ENDS:
Produced when each of the strands extends beyond the complementary region of the strand pair.

Question 6

Ligases

Answer 6

JOIN DNA

• Catalyse the joining of pieces of double-stranded DNA at there sugar-phosphate backbones.

Question 7

What does CRISPR-CAS9 stand for

Answer 7

Cluster Regularly Interspaced Short Palindromic Repeats - CRIPSPR -associated protein 9.

*Used to fight against bacteriophage.

Question 8

Describe the 5 steps of CRIPSPR-Cas9 as a defence mechanism.

Answer 8

STEP ONE:
- Bacteriophage injects its DNA into a bacterium cytosol. (identified as foreign)
- Cas 1 & 2 find the PAM site (xGG) and cut approx. 30 nucleotides upstream from the PAM site out from the DNA sequence. (-> Then becomes protospacer while it its being transported by CAS 1&2)
- Protospacer then incorporated into CRISPR gene locus = Spacer.

STEP TWO:
- The CRIPSR array is transcribed into a single pre-crRNA molecule. TracrRNA binds to the repeats int eh CRISPR array. RNAse enzymes cut the pre-CRIPSPR to make cr:tracrRNA, which becomes gRNA when it is picked up by a Cas9 enzyme.

STEP THREE:
- Armed with gRNA, Cas9 searches DNA for a PAM, and upon finding it, scans for invading bacteriophage DNA that is complementary to its gDNA.
- The complex with cur both template & coding strand of virus = blunt ends.
- Viral DNA is no longer active (if repair mechanisms of cell fixed he DNA, the extermination step 3 will occur again)

Question 9

Steps of obtaining CRIPSR-CAS9 in gene editing

Answer 9

1. Single guide RNA (sgRNA) created that is complementary to target zone.
2. Cas9 protein obtained & mixed with sgRNA
3. Mixture injected into specific cells.

Question 10

How gene editing with CRISPR works

Answer 10

1. TARGETING: Cas9 sgRNA complex searches for a PAM site
2. BINDING: sgRNA is complementary to target gene & binds
3. CLEAVAGE: The Cas9 protein (type of Endonuclease) cuts the DNA 2-6 base pairs upstream from PAM site.
4. DNA REPAIR: Could add or delete nucleotides causing a frame shift to render the gene non-functional

Question 11

What are future applications of CRISPR?

Answer 11

RESEARCH:
- e.g., Attach fluorescent protein to cas9 to locate a specific gene in genome.
- e.g., Disrupt expression of gene to see effect of not having that protein. -> helps identify function of specific genes.

DEALING WITH DISEASES:
- e.g., Replacing deleterious allele with healthy
- e.g., Adding genes that code for proteins that decrease susceptibility to infectious diseases.

AGRICULTURE:
- e.g., Introducing pest & herbicide-resistant genes to increase yield of crops.

Question 12

Limitations of using CRISPR-Cas9 in gnene editing

Answer 12

• Elimination of genetic diseases has been possible in animal models but not humans.
• To knock-in a new segment of DNA it requires chance that the inserted DNA will be accepted by repair machinery, so low precision rates.
• Ethical implications
  -> To successfully alter an organisms genome using CRISPR-Cas9, scientits must use an embryo to get DNA info.

Question 13

risks of CRIPSR on humans

Answer 13

• short sequences by the sgRNA may be repeated many other times so it could be off target
• DNA repair after the CRIPSR-CAS9 action may not occur
• CRISPR CAS9 may cut out functional genes needed for the cell.

Question 14

production of insulin

Answer 14

restriction site cut with endonuclease and same spot in the subunit gene (a&b).

• sticky ends join to sticky ends of restriction site using ligase
• transformation of vacteria with plasmids and expression of proteins (heat shock or electroporation)
• extracts proteins and combine to create insulin