BCHE 545/MolB 545

Biochemical and Molecular Genetics

Spring Semester, 2011
Tue/Thur 8:55 – 10:10 AM, Foster Hall Room 146
Instructor: Jennifer Curtiss
 Part II & III
• Tools for Studying Genes and Gene Activity
• Genomics/Transcriptomics/Proteomics

Instructor: Immo Hansen

 Flow of genetic information:
• In cells:
DNA – RNA – Protein
• In retro viruses:
RNA – DNA – RNA - Protein
 Molecular Cloning
• What is a clone?
– a group of identical cells or organisms

• Gene cloning:
– put a foreign gene in bacterial cells (E. coli) and
grow many of them
– production of a large number of identical DNA
molecules
Example: Cloning of GFP in E. coli
The Holy Book of Molecular Cloning:
 1. Agarose gelelectrophoresis
• a widely used technique for the analysis of
nucleic acids
• separates molecules on the basis of their rate
of movement through a gel under the
influence of an electrical field
• Determine presence and size of a given DNA
molecule
 Agarose gelelectrophoresis
• DNA is negatively charged due to phosphates
in its backbone and moves to anode, the
positive pole
• When placed in an electrical field, DNA will
migrate toward the positive pole (anode)
• An agarose gel is used to slow the movement
of DNA towards the anode and separate by
size
 Agar
• mixture of agarose and agaropectin

Youkan mizuyoukan
Agarose
 Agarose gelelectrophoresis
– Small DNA pieces have little frictional drag so move
rapidly
– Large DNAs have more frictional drag so their mobility
is slower
 Agarose gelelectrophoresis

H O2
 

DNA

- +

Power
How fast will the DNA migrate?
strength of the electrical field, buffer, density of agarose gel…
Size of the DNA!
*Small DNA move faster than large DNA
…gel electrophoresis separates DNA according to size

DNA

small
large

- +

Power
Within an agarose gel, linear DNA migrate inversely
proportional to the log10 of their molecular weight.
 Results
• DNA distributes according to size
• Largest near the top
• Smallest near the bottom
• DNA is stained with fluorescent dye

5-16
2. Restriction Endonucleases
 Restriction Enzymes (Type II)
• enzyme that cuts double-stranded DNA at
specific recognition sequences known as
restriction sites
• found in bacteria and archaea
• biological function is to protect cells from
foreign DNA
 Nomenclature of
restriction enzymes
• EcoRI: E coli, R strain, 1st enzyme found
• PstI: Providencia stuartii, 1st enzyme found
• NotI: Norcardia otitidis-caviarum
• How would you call the third restriction
enzyme isolated from Haemophilus influenza
strain D?

• HinDIII
How long is the recognition sequence?

• 4 bp : e.g., Taq 1, HpaII, MspI

• 6 bp : e.g., EcoR1, HindIII, BamH1, PstI, SalI
• 8 bp : Not I, Sfi I
Recognition Site is palindromic

OTTO
 Recognition sequence may be
interrupted or ambiguous

• Acc I: GT(at/gc)AC

• Sfi I: GGCCNNNNNGGCC

• Afl III: ACPuPyGT

Three types of ends produced by
type II restriction enzymes

• 3’-overhang (protruding)
• 5’-overhang
• blunt end
 5’-overhang
5’-----------------------gaattc---------------------------3’
3’-----------------------cttaag--------------------------5’

X EcoR1

5’-----------------------g + 5’-aattc---------------------------3’
3’-----------------------cttaa -5’ g---------------------------5’
 3’-overhang
5’-----------------------ctgcag---------------------------3’
3’-----------------------gacgtc--------------------------5’

X PstI

5’-----------------------ctgca-3’ 5’-g---------------------------3’
3’-----------------------g-5’ 3’- actgc---------------------------5’
 Blunt end

5’-----------------------gatatc---------------------------3’
3’-----------------------ctatag--------------------------5’

X EcoR V

5’-----------------------gat-3’ 5’-atc---------------------------3’
3’-----------------------cta-5’ 3’-tag---------------------------5’
 Selection of restriction
enzymes
• Of Over 250 commercially available and
• over 2,000 total, how do I know what to use?
• Cutting frequency
• Easy to work with
• Economical
 3. DNA Ligases
• forms covalent
phosphodiester bonds
between 3' hydroxyl
ends of one nucleotide
with the 5' phosphate
end of another
• ATP is required
DNA Ligase connects two sticky ends
 4. Phosphatases/polynucleotide
kinases
• Alkaline Phosphatase removes phospho
groups from 5’-ends of DNA
• Prevents autoligation
 T4-Polynucleotide kinase
• catalyzes the transfer of a gamma phosphate
from ATP to the free hydroxyl end of the 5'
DNA
 Vectors
• function as DNA carriers to allow replication of
recombinant DNAs
• There are 2 major classes of vectors:
– Plasmids
– Phages

4-32
5. Plasmids
 What is a plasmid?
• An extra-chromosomal DNA (genome) molecule
• Replicates in cytoplasm of bacteria or in nucleus of eukaryote
(autonomous replication)
• Double-stranded, circular
• Found in bacterial cells
• Carry a few genes; small = few thousand bps
• Independent of the bacterial chromosomal genome
• Have a single origin of replication
• Replicated by same cell machinery as the bacterial
chromosome
• Basis of recombinant DNA technology
 Plasmid transfer
• Host-to-host transfer:
– Direct transfer by conjugation
– Transformation (uptake of the
plasmid)*

Function of a plasmid in a bacterial cell:

-carry antibiotic resistance genes
-may help the bacteria to fix nitrogen or
degrade organic compounds (in
conditions of nutrient deprivation)
 Electron micrograph of an E. coli cell ruptured to release its
DNA. The tangle is a portion of a single DNA molecule containing
over 4.6 million base pairs encoding approximately 4,300 genes.
The small circlets are plasmids. (Courtesy of Huntington Potter
and David Dressler, Harvard Medical School.)

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RecombinantDNA.html
Why do we use them in molecular biology?
• Easy to manipulate and isolate using bacteria

Used to:
-Integrate a gene function into a mammalian
cell/organism (transient transfection)
- produce a recombinant protein for human therapy
- Study genes and manipulate it
 Two categories of plasmids:
1) Stringent plasmids – replicate only when the
chromosome replicates; once per cell division
2) Relaxed plasmids – replicates independent of
the chromosome; these are used in molecular
cloning experiments
-replicate regardless of cell division
 Plasmid requirements
• Origin of Replication (ORI)

• Selectable marker
(such as antibiotic resistance gene)

• Restriction enzyme sites placed

in non-vital areas
multiple cloning site (MCS)
 pBR322 Plasmid
• pBR322 illustrates cloning
methods simply
– Resistance for 2 antibiotics
• Tetracycline
• Ampicillin
– Origin of replication
between the 2 resistance
genes
– Only 1 site for several
restriction enzymes

4-40
 pBR322 Cloning
Clone a foreign DNA into the
PstI site of pBR322
• Cut the vector to generate
the sticky ends
• Cut foreign DNA with PstI
also – compatible ends
• Combine vector and foreign
DNA with DNA ligase to seal
sticky ends
• Now transform the plasmid
into E. coli
4-41
 Bacterial Transformation
• Traditional method involves incubating
bacterial cells in concentrated calcium salt
solution
– The solution makes the cell membrane leaky,
permeable to the plasmid DNA
• Newer method uses high voltage to drive the
DNA into the cells in process called
electroporation

4-42
 b-galactosidase
Newer cloning plasmids (eg. pCR2.1-TOPO) have:
– Ampicillin resistance gene
– Multiple cloning site inserted into the gene lacZ’
coding for the enzyme b-galactosidase
• Clones with foreign DNA in the MCS disrupt the ability
of the cells to make b-galactosidase
• Plate on media with a b-galactosidase indicator (X-gal)
and clones with intact b-galactosidase enzyme will
produce blue colonies
• Colorless colonies should contain the plasmid with
foreign DNA

4-43
Blue white screen
 Directional Cloning
• Cut a plasmid with 2 restriction enzymes from
the MCS
• Clone in a piece of foreign DNA with 1 sticky
end recognizing each enzyme
• The insert DNA is placed into the vector in
only 1 orientation
• Vector religation is also prevented as the two
restriction sites are incompatible

4-46
A plasmid cloning and transformation movie

• http://www.sumanasinc.com/webcontent/ani
mations/content/plasmidcloning.html
 6. DNA-dependent DNA Polymerases

• any of various enzymes catalyzing the

template-directed incorporation of
deoxyribonucleotides into a DNA chain, using
a DNA template
• Taq
• Pfu
• Vent
• Etc.
 7. Reverse Transcriptases
• RNA-dependent DNA polymerase
• Encoded by retroviruses
• Used for synthesis of DNA from RNA
templates - cDNA
 thymidine with
azide group
DNA binding
site
 Modern HIV therapy strategies
• Aim to prevent evolution of resistance
• How: Increase the number of mutations needed to make the
virus resistant
• Combine several drugs with different mode of action
– RT inhibitors
– Protease inhibitors
– Fusion inhibitors
– Integrase inhibitors
• Highly effective but strong side effects
• Does not remove virus from body
• Multidrug resistance has been reported
Highly active antiretroviral therapy (HAART)
 Next:
• Jan 26th

»PCR
– PCR principle
– Primer selection
– RACE PCR
– Real time PCR
– Sanger sequencing