32. Gene editing and DNA manipulating

Question 1

What does denaturing/melting DNA refer to?

Answer 1

Separating double stranded DNA into single strands

Question 2

What does renaturing/hybridisation refer to?

Answer 2

Means joining single stranded nucleic acids into double strands.

Question 3

What are the two molecular forces that need to be broken for the denaturing of DNA?

Answer 3

1. Hydrogen bonds between complementary bases

2. Stacking of hydrophobic Nitrogenous bases down the centre of the helix

Question 4

What are the two means by which DNA can be denatured?

Answer 4

1. High temperature, >80

2. Chemical, strong alkali

Question 5

What are the two means by which DNA can be renatured?

Answer 5

1. Low temperature, less than 80

2. Chemical, neutral pH

Question 6

What is the difference between renaturing and hybridisation?

Answer 6

Renaturing refers to DNA - DNA strands combining

Hybridisation refers to either RNA of DNA from different sources combining with another single stranded DNA

Question 7

What components are needed for PCR?

Answer 7

1. Template or original target DNA
2. Oligonucleotide primers
3. Deoxyribonucleotide triphosphates (dNTP) building blocks
4. DNA polymerase

Question 8

What are the 3 steps of the PCR reaction?

Answer 8

1. Denaturing
2. Annealing/hybridisation of primers
3. Extension/elongation by DNA polymerase

Question 9

What are the temperatures for each stage of the PCR cycle?

Answer 9

95, 45-65, 70

Question 10

What are restriction enzymes and recognition sequences?

Answer 10

• Bacteria produce restriction enzymes which can cut DNA at specific sequences
• All restriction enzymes recognise a specific base sequence in the DNA called the recognition sequence

Question 11

What type of restriction enzymes are used in the lab?

Answer 11

Type II which have a predictable recognition sequence

Question 12

What do the type 5 restriction enzymes do?

Answer 12

Cas9-gRNA complex for example, uses RNA to target specific sequences.

Question 13

What mechanisms are in place to ensure restriction enzymes do not self recognise bacterial DNA?

Answer 13

• Restriction enzymes needed to cleave incoming phage DNA
• Methyl groups at restriction sites on the bacterial DNA block the restriction enzyme and protect the bacterial DNA from being cleaved

Question 14

What DNA can the ends be joined to depending on if they are sticky or blunt?

Answer 14

• Blunt ends can be joined with any blunt end

- Sticky ends can only be joined if the base pairs sticking out match (complementary)

Question 15

What are isoschizomers?

Answer 15

Different restriction enzymes that recognise the same sequence AND cut similarly

Question 16

What are neoschizomers?

Answer 16

Different restriction enzymes that recognise the same sequence BUT cut differently

Question 17

What charge is there on DNA?

Answer 17

DNA is negatively charged due to the negative phosphate group and will migrate from negative to the positive electrode

Question 18

What are the 4 factors which the gel electrophoresis migration rate depends on?

Answer 18

• Gel concentration: movement is slower in denser gel
• Voltage across field: Slower with lower voltage
• Buffers used
• Conformation: faster rate if DNA is more compact

Question 19

How can genomic variations be detected with restriction enzymes? (RFLP)

Answer 19

• Variations in genome may lead to loss/gain of restriction enzyme sit: restriction fragment length polymorphism (RFLP)
• If an RFLP is linked (close to) a gene mutation the RFLP will tend to be co-inherited with one of the gene alleles

Question 20

What are the four components needed for gene cloning?

Answer 20

1. DNA
2. Vector to carry the different length DNA (plasmid)
3. Restriction enzymes and DNA ligase (cut/join recombinant DNA)
4. Host cell in which vectors replicate (bacteria)

Question 21

Why are bacterial plasmids chosen as the most commonly used vector for gene cloning?

Answer 21

1. Origin of replication
2. Antibiotic resistance for selection
3. Multiple cloning sites containing unique restriction enzyme sites

Question 22

How is the host cell treated to increase uptake of recombinant DNA?

Answer 22

Transformation involves heat shock at 42 degrees to make the membrane porous to uptake DNA and electroporation

Question 23

What are two ways in which host cells can be screened to see which ones have taken up the plasmid?

Answer 23

• Antibiotic resistance screening: only hosts containing the vector can grow
• LacZ screening only hosts containing the vector can make blue colonies