4 - Molecular Biology

Question 1

molecular biology

Answer 1

techniques used to map DNA

Question 2

techniques

Answer 2

• cutting
• pasting
• copying

Question 3

restriction endonuclease

Answer 3

• cuts middle of DNA
• homodimers, therefore they can only cut palindromes
• evolve to cut a specific part of DNA, but it won’t be able to cut the newly formed DNA

Question 4

sticky ends

Answer 4

• DNA dissociating after being cut

- can come together again if cooled

Question 5

blunt ends

Answer 5

no overhang on the DNA cut

Question 6

process of ligation

Answer 6

• NAD+/ATP cofactor is used to make a good LG in the E+
• the Nu- is able to attack the E+ with the DNA ligase
• sticky ends form non-covalent complex, then ligase will remake DNA and stick it back together

Question 7

blunt end ligation vs. sticky end ligation

Answer 7

blunt end is much less efficient

Question 8

plasmids

Answer 8

can be copied and amplified many times

Question 9

plasmid contents

Answer 9

• circular DNA
• antibiotic
• ORI
• restriction sites

Question 10

restriction sites

Answer 10

plasmid will contain a lot to give options for experimenters

Question 11

plasmid replication

Answer 11

1. insert DNA fragment into plasmid vector using restriction sites
2. mix E. coli with plasmid (heat pulse/electrical shock)
3. plate them in Petri dishes with ampicillin
4. transformed cells will survive
5. replicate plasmids that survived
6. replicate cells that survived (plasmid + E. coli)

Question 12

purification of plasmid DNA

Answer 12

• DNA sticks to silica really well
• mixture of silica with plasmid vector and DNA will separate the two into plasmid and DNA + silica
• adding water to DNA + silica mixture will separate the two and give pure DNA

Question 13

polymerase chain reaction (PCR)

Answer 13

• most powerful techniques used
• uses 2 primers, therefore the amplification is exponential
• can be used to add restriction sites to the ends of a gene

Question 14

ligating PCR products

Answer 14

• complementary restriction sites
• blunt edges
• TA overhangs

Question 15

Gibson Assembly Cloning

Answer 15

• inserting DNA into plasmid without restriction sites
• requires T5 exonuclease
• can combine many fragments at once
• all happens in one step
• T5 exonuclease, DNA polymerase, and ligase all in one solution

Question 16

T5 exonuclease

Answer 16

digests 5’ of DNA, leaving 3’ overhangs

Question 17

Gibson Assembly Cloning process

Answer 17

1. add overlap to one fragment with PCR
2. use T5 exonuclease to digest 5’ ends, forming sticky ends
3. recombine with non-covalent bonds
4. use PCR to close gaps in fragments
5. use ligase to close nicks

Question 18

insert restriction site in DNA

Answer 18

1. do two separate PCRs with the restriction site
   - primer will have the restriction site
2. digest and ligate

Question 19

problems with inserting restriction site with PCR

Answer 19

they can ligate to the other DNA strand through non-covalent bonds

Question 20

site-directed mutagenesis

Answer 20

1. do two separate PCRs with the mutation
   - primer will have the mutation
   - mutation will lower temperature as its base cannot pair with other strand
2. cool/lower the temperature
3. add DNA polymerase for strands that cannot extend 5’ to 3’
4. add outside primers and perform PCR to amplify desired mutation

Question 21

random mutagenesis

Answer 21

doing a PCR reaction with manganese ion (instead of magnesium ion) and low concentration of one or more dNTPs will make errors within DNA

Question 22

quickchange

Answer 22

• linear amplification, therefore NOT PCR

- most popular method of site-directed mutagenesis

Question 23

quickchange process

Answer 23

1. denature plasmid
2. add primers with desired mutation
3. add DNA polymerase until it hits nicks
4. treat with Dpn1

Question 24

Dpn1

Answer 24

endonuclease that will only digest the template DNA because only the template DNA is methylated

Question 25

methylation of DNA from E. coli

Answer 25

methylate the amine of adenine

Question 26

dam methylase

Answer 26

adenine N6 methyltransferase