A student was given two samples, A and B.

She took 3 l of sample A and added 7 l

of water and 2 l of sample buffer to it. That entire sample of 12 l was loaded into
lane 1 on the gel below. She took 3 l of sample B and added 7 l of water and 2 l of
sample buffer, and loaded that entire sample into lane 2.

1
23.1 kb
9.3 kb
6.6 kb

Amount of DNA in Hindlll bands:

23.1 kb 240 ng
9.3 kb
95 ng
6.6 kb
70 ng
2.3 kb
25 ng

1. What is the approximate amount of DNA in the band

in lane 1? ____________

4.4 kb
2.3 kb
2.0 kb

2. What are the size of the DNA fragments in lane 1?

____________
3. What is the size of the DNA fragments comprising
the DNA band in lane 2? ____________
4. What is the concentration of DNA in sample B?
____________

23,130 bp, 240 ng

9,416 bp, 95 ng
6,557 bp, 70 ng
4,361bp na
2,322 bp, 25 ng
2,027 bp, 20 ng

Which band contains more

DNA? Which band is
larger?
If I told you that the
starting concentration of
the DNA sample in lane 3
was 50 ng/ul, how many ul
of sample were loaded?

Lecture 3
Cloning DNA
Making a genomic library
Isolation of DNA from Vibrio fischeri

Lecture/Lab 3 Learning Objectives:

By the end of lab today, you will be able to
Be able to briefly explain to a non-expert the
importance of the development of recombinant DNA
technology.
Explain the function of each reagent used during
genomic DNA isolation and what bacterial component
it acts on.
Predict (generally) what would happen if a particular
reagent or step was skipped during DNA isolation.

DNA Cloning
If you introduce a foreign piece of DNA into a
another organism, and that piece of DNA gets
copied when the host cells replicate, you will
have a large number of cells all with identical
copies of that piece of foreign DNA so you
have cloned that piece of DNA, and can use
the host cells to replicate it.
If that piece of DNA codes for a protein, it is also
possible for the host cells to produce the protein.

What do you need to clone DNA?

Source of DNA to be cloned

genomic DNA or cDNA (DNA copied from RNA)
Vector to get foreign DNA into host organism
Plasmid, virus, phage, etc
Type of vector you use depends on host
Host organism
Can be bacteria, yeast, mammalian cell, etc
Bacteria easiest to use but there are sometimes
issues expressing eukaryotic proteins in bacteria

Figure 1.1The basic steps in gene cloning.

Gene Cloning and DNA Analysis by T.A. Brown. 2006 T.A. Brown.

Why clone DNA?

Study function of proteins etc. in cells.
Produce human proteins to use as drugs insulin,
growth hormone
Make genetically modified plants that are resistant
to pests or produce vitamins, etc
Gene therapy transfer good copy of a gene into a
patient with abnormal copy

A chain

Example: Cloning of human insulin

Insulin is a small protein consisting of an A chain of 21 amino

acids linked by two disulfide (SS) bridges to a B chain of 30
amino acids. It is produced by beta cells in the pancreas, and is
necessary for glucose uptake into certain tissues like liver, muscle
and fat.
Type 1 diabetics, are absolutely dependent on insulin since their
beta cells no longer secrete insulin, and even Type 2 diabetics
may become dependent on insulin.

B chain

 Prior to the production of insulin by recombinant DNA

techniques, diabetics were injected with insulin
isolated from cows and pigs.
In the 1970s,several groups attempted to identify and
then clone the human insulin gene. Genetech
however, won the patent, licensed it to Eli Lilly, who
began producing human insulin in E. coli =
Humulin
Recombinant DNA technology has also made it
possible to manufacture slightly-modified forms of
human insulin that work faster (Humalog and
NovoLog) or slower (Lantus) than regular human
insulin.

Other recombinant proteins

Human growth
hormone

Dwarfism

Factor Vlll

Blood clotting

Hepatitis B vaccine
Anti-TNF alpha
receptor antibody

Autoimmune diseases

Golden rice

Vitamin A deficiency

Chymosin

Cheese manufacturing

Creating a genomic library

A Genomic library is a collection of clones representing
the entire genome of an organism
1. Isolate genomic DNA from source
2. Break up large pieces of chromosomal DNA into smaller pieces
vectors have limits in terms of sizes of insert
3. Cut open vector, and then ligate pieces of genomic DNA into
vector
4. Transform bacteria or other host cells with recombinant vectors
to get individual clones = host cells that contain a single vector
with the same piece of DNA
5. Different clones will contain different pieces of DNA if different
pieces of DNA in all clones assembled together, the whole
genome of the organism can be recapitulated

Figure 1.3 Cloning allows individual fragments of DNA to be purified.

Gene Cloning and DNA Analysis by T.A. Brown. 2006 T.A. Brown.

Creating a genomic library from

Vibrio fischeri DNA
1. Isolate genomic DNA from bioluminescent bacteria Vibrio
fischeri
2. Digest genomic DNA with restriction enzyme SalI to create
smaller DNA fragments
3. Ligate genomic DNA fragments into plasmid vector pGEM that
has also been digested and cut open with SalI
4. Transform E. coli strain DH5alpha with recombinant plasmids
and then culture on agar-ampicillin plates
5. Select individual clones to sequence inserts in plasmids
and
6. Screen clones to see if any are producing proteins involved in
bioluminescence

Isolation of genomic DNA

1. Grow Vibrio fischeri cells in high-salt liquid media at room
temperature overnight then centrifuge to pellet cells
2. Lyse cells open
V. fischeri are gram-negative bacteria require several steps to break
open because of cell envelope

3. Inhibit DNases use EDTA, pH 8.0

4. Get rid of all other components
1. Proteins/lipids/sugars phenol/chloroform extraction
2. RNA use RNase

5. Concentrate DNA using ethanol

Bacterial
chromosome

Gram negative bacteria cell envelope

Nucleases

Nucleases are enzymes that either cut or shorten DNA or RNA

strands by breaking the phosphodiester bonds
Exonucleases remove nucleotides from the ends of DNA molecules

Endonucleases cut DNA in the middle of the chain

-Some endonucleases like DNase l are non-specific will cut DNA anywhere
in sequence
-Others, like Type ll restriction enzymes, will only cut DNA at specific
sequences
Both types of DNA nucleases require Mg2+ as a cofactor

Inhibit DNases

V. fischeri has lots of periplasmic nuclease activity so it is important

to get rid of these proteins before they have a chance to digest our
DNA!
As you break open the bacteria, the DNases that are normally
localized in the periplasmic space are in contact with
chromosomal DNA and could digest it

So start DNA purification procedure by re-suspending cells in TES

buffer:
T = 10 mM Tris buffer, pH 8.0 - DNA more stable at slightly alkaline pH
E = 1 mM EDTA to remove Mg2+ and inhibit DNases.
S = sodium chloride need sodium for ethanol precipitation later in
procedure

Lysing gram negative bacteria

1. Add a chelating agent of divalent metals (e.g. EDTA) to disrupt
outer membrane lipopolysaccharides.
Mg2+ is bound to LPS (lipopolysaccharide layer) and stabilizes
it. EDTA is a chelator that removes Mg2+ and destabilizes
membrane.
2. Add lysozyme to break up peptidoglycan layer, which is critical for
maintaining shape and rigidity of bacteria.
(Peptidoglycan layer made of polysaccharides and amino acids
lysozyme cleaves sugar component.)
3. Detergent SDS added to help lyse the cells by disrupting lipid bilayer
also denatures some proteins

Removing other cellular components

Add RNases enzymes that digest RNA (lab 4)
Add proteases enzymes that digest proteins (lab 3 & 4)
Proteinase K is actually more active in the presence of SDS, which
denatures most proteins, and is more active at higher temperatures
(up to 65C).

Phenol-chloroform extraction: both are organic solvents that

will dissolve lipids and dissolve and denature proteins.
Phenol equilibrated to pH 8 will cause lipids, protein, and
polysaccharides to partition into organic phase also dissociates
proteins from nucleic acids.
Chloroform helps the two phases separate better and is easier to
evaporate than phenol.

Figure 3.6 Removal of protein contaminants by phenol extraction.

Gene Cloning and DNA Analysis by T.A. Brown. 2006 T.A. Brown.

http://bitesizebio.com/articles/the-basics-how-phenol-extraction-works/

Concentrate DNA
Usually use alcohol to precipitate nucleic acids
out of solution
Nucleic acids have limited solubility in alcohol
solvents.
ETOH is volatile and the pellets can easily be dried.
will only work in presence of monovalent cation
monovalent cations shield negative charges and
reduce repulsion DNA precipitates.
http://bitesizebio.com/articles/the-basics-how-ethanol-pre
cipitation-of-dna-and-rna-works
/

What reagents will we deploy to disrupt the cell wall

and the membranes of these bacteria?
Discuss with your group
1.
2.
3.
4.

___________
___________
___________
___________

Explain to your nearest neighbor

1. How will you purify DNA from proteins
and lipids. Indicate the roles of the
reagent(s) youll use.
2. How will you concentrate your DNA.
Indicate the roles of the reagent(s) youll
use.