Lecture 4 Restriction Enzymes and Molecular Cloning

Question 1

Cloning

Answer 1

A collection of molecules or cells, all identical to an original molecule or cell
Make a copy a piece in a plasmid or a whole organism
Ex. Twins are clones

Question 2

2 very different examples of cloning

Answer 2

1. Cell based DNA cloning

2. Cell/organism cloning

Question 3

Cell based DNA cloning

Answer 3

Cutting a piece of DNA from one organism and inserting it into a vector where it can be replicated by a host organism

Question 4

Cell/organism cloning

Answer 4

Using nuclear DNA from one organism to create a second organism with the same nuclear DNA

Question 5

Purpose of cloning?

Answer 5

Determine the sequence of recombinant DNA
To generate a probe for hybridization (Southern blot, Northern blot, etc)
Recombinant expression of the encoded protein and mutagenesis
Regenerative medicine - transplant

Question 6

Restriction Endonucleases

Answer 6

Restriction Enzymes
Enzymes that cleave DNA molecules at specific nucleotide sequences, called restriction sites
1962 “molecular scissors” discovered in bacteria by Nathans and Smith in 1970
E. coli bacteria have an enzymatic immune system that recognizes and destroys foreign DNA

Question 7

Restriction Sites

Answer 7

Where enzymes cleave DNA molecules at specific nucleotide sequences
3,000 enzymes have been purified, around 200 have unique properties

Question 8

General info about restriction endonucleases

Answer 8

Most are 4-8 base pairs
Isolated from bacteria, different types of bacteria so different restriction enzymes
Enzymes that will cut DNA at specific sites defined by the local nucleotide sequence, cleaving a dsDNA into fragments of defined sizes

Question 9

Most common Restriction Enzyme

Answer 9

EcoRI

Escherichia coli strain R, 1st enzyme

Question 10

3 Types of Restriction Enzymes

Answer 10

Differ in their structure and whether they cut their DNA substrate at their recognition site, or if the recognition and cleavage sites are separate from one another.
Type I - cuts at random locations
Type II - cut DNA within the recognized sequence without need of ATP
Type III - cuts at approximately 25 base-pairs from the site and also requires ATP (very rare)

Question 11

Restriction Enzyme Type I

Answer 11

Cuts DNA at random locations as far as 1000 or more base-pairs from the recognition site and requires ATP
-random

Question 12

Restriction enzymes Type II

Answer 12

Cut DNA within the recognized sequence without the need of ATP
-most used, common because they cut at the recognized restriction site or adjacent

Question 13

Restriction enzyme Type III

Answer 13

Cuts at approximately 25 base-pairs from the site and also require ATP
-very rare

Question 14

How Type II Restriction Enzymes work

Answer 14

Cut in close proximity of the recognition site
No ATP requirement
Need Mg2+ as cofactor (in buffer)
Recognition sites in double stranded DNA have a 2 fold axis of symmetry - a palindrome
Cleavage can leave stick end or blunt ends
More than 3500 REs
Most useful for gene analysis and cloning

Question 15

RE

Answer 15

Restriction Enzymes

Question 16

Sticky Ends

Answer 16

Also called cohesive since they are easily joined back together by a ligase
A nucleotide fragment can be easily joined with an sticky end vector if digested with the same RE
Allow to do unidirectional cloning

Question 17

Blunt End Digestion

Answer 17

Vs. Sticky ends
Always compatible with each other
-harder to ligate, less efficient
Disadvantage of potentially inserting the insert DNA in the opposite orientation desired

Question 18

Plasmids

Answer 18

Naturally occurring extrachromosomal DNA molecules
Circular, double stranded DNA
Can be cleaved by restriction enzymes (sticky or blunt ends)
Can be engineered by linking new DNA fragments to the ends of plasmid
Carries antibiotic resistance cassete and the resistance is often transferred from one bacteria to another

Question 19

Ori

Answer 19

Origin of replication

Question 20

Steps of Recombinant DNA

Answer 20

Cut plasmid vector with Aval
Excise DNA insert of interest from source using Ava I
Ligate the insert of interest into the cut plasmid

Question 21

4 Things important for Cloning Vectors

Answer 21

1. Origin of replication - where gene starts
2. A selectable marker - antibiotic resistance gene, such as amp^r and ter^r - will grow in medium that has ampicillin and tetracycline
3. Multiple cloning site (MCS) - site where insertion of foreign DNA will not disrupt replication or inactivate essential markers
4. Might carry a tag (MYC, HA, Flag) or a marker (eGFP)

Question 22

Origin of replication

Answer 22

Useful for cloning vectors

-where gene starts

Question 23

Selectable marker

Answer 23

Antibiotic resistance gene on plasmid

Question 24

Multiple cloning site (MCS)

Answer 24

On plasmid, where insertion of foreign DNA will not disrupt replication or inactivate essential markers

Question 25

Ex of tags used by plasmids

Answer 25

MYC, HA, Flag

or a marker eGFP

Question 26

Ligation

Answer 26

The process of joining two pieces of DNA from different sources together through the formation of a covalent bond
DNA ligase is enzyme used to catalyze this reaction
DNA ligation requires ATP

Question 27

DNA Ligase

Answer 27

The enzyme used to catalyze the reaction of joining two pieces

Question 28

Ligation of Complementary Ends

Answer 28

Enzymes that cut with staggered cuts result in complementary ends that can be ligated together as long as they are complementary
DNA fragments with blunt ends generated by different enzymes can be ligated together (with lower efficiency) but usually cannot be re-cut by either original restriction enzyme
If only one kind of restriction site is used the insert can connect with vector in two orientations
Vector can reconnect with itself without an insert - self ligation

Question 29

Directional Cloning

Answer 29

Directional ligation requires that two different restriction sites are used at the ends of insert and vector

Question 30

Identification of Clones and Insert

Answer 30

The clones are evaluated for the presence and the size of an insert and clones with appropriate inserts by using restriction enzymes that cut at sites flanking the insert or within the insert (if known) and gel electrophoresis

• Uncut gel there are bands higher up and make frowny face
• BamHI Digest

Question 31

Bacteria Transformation

Answer 31

The process of introducing the foreign DNA into the bacterial cell is called transformation
Bacterial act as a host cell so the DNA can be replicated
Not every bacterial cell is able to take up plasmid DNA
Bacterial cells that can take up DNA from the enviro are said to be competent
Two methods for transforming: heat shock and electroporation

Question 32

Competent

Answer 32

Bacterial cells that can take up DNA from the environment are said to be this

Question 33

Two methods for transforming

Answer 33

1. Heat shock

2. Electroporation

Question 34

Expression in Mammalian Cells

Answer 34

To produce many copies of a particular DNA sequence, the fragment is first inserted into a plasmid vector. The resulting recombinant plasmid DNA is then introduced into a bacteria, where it can be replicated many millions of times.
Genomic DNA is isolated and purifiied - plasma prep DNA isolation

Question 35

How are Plasmids Transferred into Mammalian Cells?

Answer 35

1. Nucleofection (electroporation) -electrical conductivity changes the permeability of the cell membrane
2. Lipofection (calcium chloride, chemical) -chemical that takes advantage of the liposomes. Make holes in cell that allow transfection, not very efficient
3. Viral Transduction
   - Adenovirus - very effective for high level transient expression. Not stable
   - Retroviruses - Very effective for stable expression, only low level expression

Question 36

Expressing your cloned gene

Answer 36

Even if your plasmid contains insert, it may not be able to generate functional protein from your cloned DNA

• gene may not be intact or mutations that could have been introducted
• the protein encoded by gene may require post-translational modifications
• some enzymes are a complex of peptides expressed from separate genes

Question 37

Dolly the Sheep

Answer 37

Sheep cloned by nuclear transfer from a cultured cell line

Question 38

Regenerative Medicine

Answer 38

Bone - osteoblasts
Skin
Nervous system
Liver cells
Pancreas - insulin producing cells
Heart - cardiomyocytes
Vessels - endothelial cells
Blood cells
Kidney