Cloning and Restriction Mapping

Question 1

How did we create insulin using molecular cloning?

Answer 1

We got an expression plasmid and cut the plamsid with restriction enzymes
Step 2- We obtained the humna insulin gene using cDNA (extract mrna-convert to cDNA, amplify, and cut with restriction enzyme)
Step 3- We inserted the cDNA into the expression plasmid
Step 4- We put the plamsid in bacteria and let it make the protein

Question 2

What are restriction enzymes? What are restriction sites?

Answer 2

These are enzymes originally isolated from bacteria, where they cut up invading viruses, they cleave phosphodiester bonds at specific DNA sequences called restriction sites.

Question 3

What does it mean that restriction sites are usually palindromic?

Answer 3

Are read the same from 5’ to 3’ in both strands.
ex:
5’ CGAAAA 3’
3’ AAAAGC 5’

Question 4

What kind of ends do restriction enzymes produce?

Answer 4

Staggered ends (have overhangs)
blunt ends (no overhangs)

Question 5

Fragments produced by a restriction enzyme digest are called?

Answer 5

restriction fragments

Question 6

Can you join pieces of DNA cut with the same restriction enzyme? What occurs in the newly ligated DNA?

Answer 6

Yes, through DNA ligase, you see the exact same restriction site sin the originals

Question 7

How do you ligate blunt ends?

Answer 7

Any pieces of DNA with blunt ends can be ligated

Question 8

How does the ligation efficiency change for blunt ends and sticky ends?

Answer 8

sticky ends is higher and the help efficiate gluing

Question 9

How do you determine which end is overhanging?

Answer 9

Whatever side the overhang ends closest to 5’ or 3’ end

Question 10

What does it mean to use single enzyme cloning to combine insert DNA with vector DNA?

Answer 10

You use one type of enzyme for ex: ECORI to cut both the vector dna and cdna, then you anneal both by ligase into the plasmid

Question 11

In single enzyme cloning how can insertion happen?

Answer 11

In either orientation

Question 12

How does double enzyme cloning work? How can the different pieces of dna be inserted into the plasmid (vector dna)

Answer 12

You use two enzymes to cut both the cdna and plasmid vector dna, can only be inserted in one orientation as not the same cutsites.

Question 13

What are the three essential features of cloning vectors?

Answer 13

The origin of replication
The antibiotic resistance gene
the polylinker (also called the multiple cloning site)

Question 14

What is the origin of replication (the ori)?

Answer 14

it allows plasmid to replicate in the bacterial host cells

Question 15

What is the antibiotic resistance gene?

Answer 15

allow only cells with
the plasmid to grow so it forces bacteria to keep the plasmid

Question 16

What is the polylinker?

Answer 16

is a region that contains restriction sites, this is the only region that has them and restriction sites are in a very specific order

Question 17

What are optional features of cloning vectors? Means we don’t put them in

Answer 17

Promoter systems (required for expression)- allows either constitutive or inducible expression of the gene insert
Promoters
3’ UTR, 5’ UTR, Shine dalgarno sequence and terminator sequences

Question 18

Where is promoter found in relation to the polylinker?

Answer 18

Is found upstream

Question 19

Where is the gene inserted in the vector plasmid?

Answer 19

In the polylinker

Question 20

How do we obtain the gene of interest (what we’re inserting)?

Answer 20

two ways:
DNA library
PCR

Question 21

What template DNA do we use for the PCR to get the gene we want? Why?

Answer 21

mRNA, this is because it has introns cut out

Question 22

What do we convert mRNA to before we insert it?

Answer 22

cDNA

Question 23

What do we do if the cDNA doesn’t have convenient restriction sites?

Answer 23

We add them during PCR

Question 24

How do we add restriction sites during PCR?

Answer 24

We add restriction site son primers 5’ ends which will be added after rounds of PCR

Question 25

Why can we just add restriction sites to the 5’ end? Why not the 3’ end?

Answer 25

Cause we need the 3’ end to anneal to build off of, 5’ end can flap around

Question 26

What is the purpose of restriction mapping?

Answer 26

Checking if what we did was right

Question 27

What can restriction digests help us determine?

Answer 27

The orientation of insertion
That the expected dna was inserted