Lecture 2

Question 1

What are PCR rxns catalyzed by?

Answer 1

Polymerases
Generally use dna as template

Question 2

All reactants needed for pcr

Answer 2

dNTPs
Polymerase
Primers
DNA template
Buffer w Mg2+

Question 3

Why is Mg2+ needed for pcr

Answer 3

Cofactor for polymerase

DNA stability

Question 4

Pcr cycle

Answer 4

Melt 98C 30s

Melt 98C 10s
Anneal 50C 30s
Extend 72C 90s

Extend 72C 10 min

Question 5

What end does polymerase extend from?

Answer 5

3 prime oh that is free

Question 6

Caveats of our study

Answer 6

-15 residues in binding protein

300 possible options for mutations

-higher order interactions ignored

-ignoring long distance effects

Question 7

How many sets of primers needed for GA?

Answer 7

2

2 ga rxns simultaneous per side of DNA

Question 8

Each primer has overhang on which of its ends?

Answer 8

5’

It’s 3’ end must be annealed and free for polymerase

Question 9

For protein expression, where in vector to put insert?

Answer 9

Promoter
RBS = ribosome binding site
ATG start

Question 10

What else is important for insert location?

Answer 10

Reading frame

Question 11

When to remove stop codon from insert?

Answer 11

?

Question 12

Is HCAII an example of convergent evolution?

Answer 12

YES

Question 13

How many isoforms of human HCAII?

Answer 13

7

Question 14

Isoform definition

Answer 14

?

Question 15

What type of enzyme is HCAII

Answer 15

Metalloenzyme

Question 16

What does zinc act as within enzyme?

Answer 16

Lewis acid

Question 17

When zn2+ is coordinated by 3 neutral histidines, what does zn have a high affinity for?

Answer 17

Hydroxyl group

Acts as a nucleophile and attacks co2 -> carbonic acid

Question 18

Steps for HCAII

Answer 18

?

Question 19

RLS for HCAII?

Answer 19

Step 6
Base coming in for protein

Question 20

Catalytic components

Answer 20

Glu 106
Thr 199
His 64

Question 21

Stability components

Answer 21

His 94, 96, 119

Question 22

Mutagenesis definition

Answer 22

?

Question 23

Conservative vs no conservative definitions

Answer 23

?

Question 24

Plasmid definition

Answer 24

?

Question 25

Components of cloning

Answer 25

Ori = origin of replication = how vector clones itself Selection marker = commonly an antibiotic resistant gene MCS - restriction enzyme site = site of snip = where insert can go ? - promoter (T7) - tags = many functions

Question 26

Gibson Assembly

Answer 26

Cloning method that links and amplifies both vector and insertb

Question 27

Roles of exonuclease, polymerase, and ligase in GA

Answer 27

?

Question 28

Forward primer

Answer 28

Matches with written out gene exactly Complement to anti sense strand

Question 29

Reverse primer

Answer 29

Reverse complement to 3’ end of written out gene Complement to sense strand

Question 30

Sense strand

Answer 30

What is written out = gene standard mRNA sequence Template for translation

Question 31

Anti Sense Strand

Answer 31

Actually encodes genetic info

Question 32

Cloning definition

Answer 32

Clone gene into vector

Question 33

Bp Range for primers

Answer 33

15-25bp Free 3’ oh

Question 35

RNA Virus and qPCR

Answer 35

?

Question 36

Generic way a gene is written out

Answer 36

5’ to 3’ Sense strand ATG