Topic 12: Cloning and Restriction Mapping

Question 1

Ligation efficiency is much —– for blunt ends than for sticky ends

Answer 1

lower

Question 2

Any piece of DNA with blunt ends can be

Answer 2

ligated together

Question 3

Molecular Cloning

Answer 3

Create recombinant DNA molecules to be replicated in cells

Question 4

Restriction enzymes (2)

originally+cleaves

Answer 4

• Enzymes originally isolated from bacteria where they cut up invading bacteriophage DNA
• Cleaves phosphodiester bonds between nucleotides at specific DNA sequences

Question 5

Palindromic

Answer 5

The same sequence is read on each strand 5’-3’

Question 6

restriction enzymes can create two types of ends:

Answer 6

staggered
blunt

Question 7

Restriction Fragments

Answer 7

fragments produced by from a restriction enzyme digest

Question 8

Steps for recombinant DNA technology

Answer 8

1. Obatin an expression plasmid
2. Obtain the human insulin gene (CDNA)
3. insert the cDNA into the expression plasmid (perform restriction mapping to confirm)
4. Introduce the rcombinant plasmid into bacteria

Question 9

Overview of Step 1/2 for recombinant DNA tech: Single enzyme cloning

Answer 9

Single ecoR1 cut site on vector and forms 2 cut sites after cDNA inserted
cDNA is cut twice at either side

Question 10

Overview of Step 1/2 for recombinant DNA tech: double enzyme cloning

+insertion only…

Answer 10

used different 2 enzymes to form 2 cut sites on vector plasmid and 2 cut sites on cDNA.
—–.
Insertion only happens in one orientation as the 2 dna sequence are different due to the diff restriction enzyme and you can’t “flip it” to insert in any directions. Can only be inserted in one way.

Question 11

Step 1: obtaining vector: Essential features of cloning vectors (3)

Answer 11

• origin of replication
• antibiotic resistance gene
• polylinker

Question 12

Orgin of replication (2)

allows+site where

Answer 12

• allows the plasmid to replicate in the bacteria host cells
• site where replictaion occur in bacteria

Question 13

Polylinker

Answer 13

site containing any unique restriction enzyme sites

Question 14

Why is ligation efficiency lower for blunt ends then sticky ends?

Answer 14

This is due to the absence of hydrogen bonding between the complementary nucleotide overhangs, which stabilizes the creation of the vector/insert complex in contrast to sticky end cloning.

Question 15

The promotor is found —– of the polylinker and the gene is inserted —- of promotor

Answer 15

1. upstream
2. into the polylinker, downstream

Question 16

Restriction maps

Answer 16

Maps of dna sequence of plasmid

Question 17

Pieces of DNA cut with the same restriction enzyme can be—- the newly ligated DNA—-

Answer 17

ligated together
kept the og restriction site

Question 18

the gene in creating a recombinant dna is inserted in

Answer 18

the polylinker region

Question 19

Step 2: obtaining gene to be inserted

Answer 19

• used pcr
• we need mRNA and then convert that to cDNA bc bacteria doesnt have intron so we need it already gone

Question 20

Step 2: obtaining gene to be inserted: Sticky ends (4)

Answer 20

• we want to insert the cDNA of interest into the vector
• If the cDNA doesnt have convient restriction sites on eother end, we can add them during PCR
• Use PCR primers with restriction sites on 5’ ends (These will be incorporated into the PCR product upon further rounds of PCR)
• Digest the PCR product

Question 21

Important that 3’ primer is….. because….

Answer 21

• perfectly complimentary , less important for 5’ end
• In contrast, the 5’ end of the primer is less critical for the initiation of DNA synthesis. The 5’ end is involved in the initial binding of the primer to the template DNA, but the actual synthesis of the new DNA strand occurs at the 3’ end in the PCR. As long as the 3’ end is complementary to the target sequence, the 5’ end can have some variations without significantly affecting the efficiency and specificity of PCR.