Restriction Enzymes/CRISPR

Question 1

What are restriction enzymes?

Answer 1

bind and cut specific DNA

are a form of bacterial immune system

Question 2

Why do restriction enzymes’ recognition sequences need to be palindromes?

Answer 2

restriction enzymes are homodimers, meaning they are composed of 2 identical subunits

2 peptide chains come together to form a quaternery protein

Question 3

How do restriction enzymes not cut the bacteria’s DNA?

Answer 3

Self-DNA is marked through DNA methylation to protect sites in the genome

This self marking is highly conserved and necessary

Question 4

DNA palindromes

Answer 4

the same sequence when read 5’-3’ on both strands

Question 5

Steps of restriction enzymes working

Answer 5

Restriction enzyme cleaves the incoming DNA phage at recognition sites and then other enzymes degrade phage DNA into smaller fragments

Question 6

How are restriction enzymes used by scientists?

Answer 6

Cut and paste DNA fragments together with restriction enzymes and ligase

cut both plasmid and the gene of interest with the same restriction enzyme

leaves single-stranded DNA that overhangs

ligase can paste the plasmid and gene together

Question 7

Sticky ends

Answer 7

ends of DNA molecules that are cut with the same restriction enzyme that are able to complementary pair and be sealed by ligase

Question 8

What has to happen to express human genes in bacteria?

Answer 8

cDNA has to be used

cDNA has intron sequences removed

Question 9

CRISPR definition

Answer 9

Clustered Regularly Interspaced Short Palindromic Repeats

short palindromic sequences of DNA

Question 10

Spacer DNA

Answer 10

found inbetween CRISPR repeats of DNA

Spacer DNA differs, but CRISPRs are the same repeats

Question 11

Is CRISPR a form of adaptive or innate immunity? Why?

Answer 11

Adaptive immunity

Spacer DNA comes from a virus that had previously infected the cell, but did not destroy the host, so the main chromosome picked up some viral DNA

This informs the host which viruses to watch out for

Question 12

What happens when a virus infects a cell that already has a similar spacer sequence?

Answer 12

a guideRNA is formed that can recruit cas-9

Question 13

What is gRNA composed of?

Answer 13

unique spacer RNA

CRISPR sequence

and a scaffold sequence that binds cas-9

Question 14

cas-9

Answer 14

a nuclease that cuts the target viral DNA is there is enough homology between gRNA sequence and the viral DNA

Question 15

What type of bonds does cas-9 cut?

Answer 15

covalent phosphodiester bonds to cut the DNA backbone

does not cut the hydrogen bonds between base pairs

Question 16

Two ways that cutting DNA with cas9 leads to genome editing?

Answer 16

1) Nonhomologous end joining: destroys the functioning of the gene
2) Homology Directed Repair (HDR): can edit/modify the genome, not just destroy

Question 17

Non-homologous end joining

Answer 17

broken DNA is joined back together, but with insertions and deletions

this disrupts gene function

alters the reading frame of coding sequences and alters regulatory sequences function

Question 18

Homology Directed Repair

Answer 18

intact segment of DNA that spans the region of the break can be exchanged for the broken DNA

add this DNA with CRISPR-cas9 technology

Question 19

What does both Non-Homologous end joing and homology directed repair rely on?

Answer 19

DNA repair mechanisms triggered by DOUBLE bond breaks

Question 20

Knockout mice definition

Answer 20

a mouse whose DNA has been genetically engineered so that it does not express particular proteins

Question 21

Chimera

Answer 21

individual that is created when cells of different genotypes come together to form an embryo

Question 22

Name of technique used to generate knockout mice

Answer 22

Reverse genetics

Question 23

Steps of reverse genetics to create mutants

Answer 23

1. DNA molecules are constructed to prevent function of a gene that is going to be mutated
2. DNA is added to embronyic stem cells
3. In some stem cells the mutation is taken up
4. Embryonic stem cells are injected into blastocysts to generate chimeras
5. Mutant chimera mice and bred with normal mice to generate either heterozygotes or normal mice
6. Heterozygotes are bred together

Question 24

When is CRISPR-cas9 used in reverse genetics?

Answer 24

Makes the embryonic cells more able to take up the mutant DNA