Topic 12 Cloning And Restiction Mapping

Question 1

What is molecular cloning

Answer 1

Creating recombinant dna molecules to be replicated in cells

Question 2

What are the steps in cloning

Answer 2

1. Obtain an expression plasmid (vector) from bacteria
   2.obtain the gene you want to clone (cDNA)
2. Insert the cDNA into the expression plasmid (glue plasmid and dna together)
3. Introduce the recombinant plasmid into Bactria so you can extract the protein the bacteria makes

Question 3

What are restriction enzymes

Answer 3

Enzyme that are originally from bacteria that cut invading bacteriophage

We use it to cleave phosphodiester bonds between nucleotides at specific dna sequences (restriction sites)

These restriction sites are usually palindromic (same sequence is read on both strands 5’-3’)

Question 4

What are sticky ends (staggered)
What are blunt ends

Answer 4

Double stranded cut that makes 5’ or 3’ overhangs
Double stranded cut that makes no overhangs (cut directly in the middle)

Question 5

What are restriction fragments

Answer 5

The fragments made from restriction enzyme digests

Question 6

If sticky ends are compatible (have same restriction cuts) what can we do to them

Can we do this with blunt ends

Answer 6

Ligate (glue) them together to make recombinant dna.

The recombinant dna recreates the original cut site

We can but it’s less efficient

Question 7

What do you do steps one and two of restriction mapping

Answer 7

Cleave the insert (dna) and the vector with the same restriction enzyme (single enzyme cloning)

When vector and plasmid are anealled This recreates two enzyme cut sites

Question 8

What is single enzyme cloning
Double enzyme

Answer 8

Single: same restriction enzyme used to cut, insertions can happening in any orientation

Double: different restroom enzymes used to cut, insertions have to happen in specific orientation

Question 9

What are the essential features of the vectors (plasmids)

Answer 9

Origin of replication (lets the plasmid replicate in bacterial cells)

Antibiotic resistance gene: (lets only the cells with the plasmid to grow, forced bacteria to keep and replicated the recombinant plasmid when place on antibiotics

Polylinker (mutiple cloning site): a region on the plasmid with many diff restriction enzyme sites. This is where the insert dna goes

Question 10

What are optional features of vectors (plasmids)

Answer 10

Promoter: required if we want to express the protein, can be constitutive (strong) promoter or inductively expression promoter

Promoter is upstream of the polylinker region

Start codon of insert should be closest to the promoter

Plasmids also can have 3’ UTR, shine dalgarno, 5’ UTR and terminator sequences

Question 11

How to we obtain the insert (gene) that we want to put in the vector?

How do we get the gene of interest

Answer 11

Use PCr to amplify the gene of interest

First we extract the mRNA of the gene, then convert the mRNA to cDNA (a dna copy of the mRNA)

Convert from mrna because mRNA doesn’t have introns and bacteria can’t cut out introns.

Then we use and amplify the cDNA

Question 12

When trying to add the cDNA to the vector, what do we do if there aren’t convenient restriction sites in the cDNA

Answer 12

We the the restriction sites during PCr so that after a few rounds of replication it becomes part of the PCR product

Only need the 3’ end of the added restriction site to anneal

Question 13

Why do we do restriction digests

Answer 13

To help determine orientation of the insertion

And that the expected dna sequence was inserted

Question 14

Restriction mapping practice

Answer 14

Okay