Molecular Biology

1
Molecular Biology

• Working with DNA

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Topics

• Genomic vs. Vector DNA
• Purifying plasmid DNA
• Restriction enzymes
• Restriction maps

3
DNA

• Genomic
• Prokaryote vs. eukaryote
• Circular or linear
• One or more chromosomes
• Extra-genomic
• Vectors
• Plasmids

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Vectors Vs Plasmids

• Vector
• DNA vehicle that allows the cloning, maintenance
  and amplification of a DNA sequence
• Plasmids
• Virus
• Chromosomes
• All plasmids are vectors
• Not all vectors are plasmids

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Plasmids

• Small circular DNA molecules maintained and
  amplified in eukaryotic or prokaryotic cells
• Amplification in bacteria
• Used as vector for cloning or expression of DNA
  of interest

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Characteristics of plasmid vectors

• Restriction sites for cloning
• Origin of replication (Ori)
• Selection marker
• Genes conferring resistance to antibiotics

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DNA Isolation

• Goals
• Isolation of DNA of interest
• Chromosomal or plasmid?
• Eliminate other components
• Chromosomal or plasmid DNA?
• Proteins
• RNA
• Chemicals
• Salts, detergents, etc.

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DNA isolation (contd)

• Cell lysis
• Cell wall and membrane
• Enzymatic
• Chemical
• Mechanical
• Isolation of DNA of interest
• Differential sedimentation
• Chromatography
• Removing other components
• Enzymatic
• Differential sedimentation
• Chromatography

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Plasmid DNA isolation by alkaline lysis (E.coli )
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Solutions Used

• Sol. I Resuspension buffer
• Tris HCl Buffer that protects nucleic acids
• EDTA - Chelates Mg, prevents nucleases from
  working
• Sol. II Lysis solution
• NaOH - pH lyses cells, denatures DNA
• SDS Dissolves membranes, denatures and binds
  proteins

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Solutions Used (Contd)

• Sol. III- Potassium acetate
• Renaturation of DNA
• Precipitates SDS
• Precipitates genomic DNA and proteins
• Isopropanol / Ethanol
• Precipitates nucleic acids (plasmid and ?)
• Salts remain soluble
• TE-RNase - Tris EDTA again RNase??

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Quantification of DNA

• Determining Conc. of DNA
• A260 of 1.0 50µg/mL or 50ng/µL
• Determining Amount of DNA
• 1mL of a solution with an A260 of 1.0 contains
  50µg DNA
• 1µL of a solution with an A260 of 1.0 contains
  50ng DNA

• Do not forget to account for the DILUTION FACTOR

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Restriction enzymes

• Endonuclease
• Cleaves internal phosphodiester linkages.
• Recognize specific double stranded DNA sequences
• Different endonucleases recognize different
  sequences
• Recognize palindrome sequences

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Palindromes

• The same sequence is read in the 5 3
  direction on both strands

5-G
G
A
T
C
C-3
3-C
C
T
A
G
G-5
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• The same phosphodiester linkages are cleaved on
  both strands!

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Different ends are generated
Blunt ends
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Different ends are generated
5 overhangs
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Different ends are generated
3 overhangs
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Compatibility of ends
Compatible
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Compatibility of ends
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Compatibility of ends
Annealing
Compatible
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Compatibility of ends
Annealing
Incompatible
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Restriction Maps
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Restriction maps

• Determining the positions of restriction enzyme
  sites
• Linear DNA maps
• Circular DNA maps (plasmids)
• Maps of inserts within vectors

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Approach

1. Determine whether the DNA has digested
2. Is the digestion complete or partial?
3. How many cuts?
4. Determine the relative positions

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Is the DNA digested?
Ladder
Control

• Compare to the undigested control
• Which samples were not digested?
• 1 and 4
• Which samples were digested?
• 2 and 3

1
2
3
4
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Is the digestion complete?

• Complete digestion
• All the DNA molecules are cleaved at all the
  possible sites
• Partial digestion
• A fraction of the molecules are not digested
• Partial undigested
• A fraction of the molecules were digested, but
  not at all the possible sites
• Partial digestion

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Complete digestion
Digestion
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Partial digestion Partial undigested
Digestion
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Partial digestion
Digestion
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Is the digestion complete or partial?

• Compare to control
• Verify the intensity of the bands
• Verify the sizes

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How many cuts?

• Number of sites
• Circular DNA number of bands
• Linear DNA Number of bands 1

33
Linear DNA maps
Enzyme Fragments (Kb)
HindIII 3 and 4
SalI 2 and 5
HindIII SalI 2 and 3
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Circular DNA maps (plasmids)
Enzyme Fragments (Kb)
BamHI 2, 3 and 5
HindIII 1 and 9
BamHI HindIII 1, 1.5, 2, 2.5 and 3
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Insertion maps
MCS
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Approach

• Determine the total size
• Determine size of the insert
• Total size size of vector
• Determine the insertion site within the MCS
• Determine which enzymes cut wihin the insert
• Relative mapping in relation to the sites at
  known positions

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Insertion maps

• Total size
• 7.7Kb
• Insert size
• 7.7 2.7 5.0Kb
• Insertion site
• Generates 2 fragments of which one is the size of
  the vector
• XbaI

Enzyme Fragments
BamHI 7.7Kb
EcoRI 1.0, 3.0, 3.7Kb
PstI 2.0 and 5.7
XbaI 2.7 and 5.0
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Insertion maps
Enzyme Fragments Total cuts Sites in vector Sites in insert
BamHI 7.7Kb 1 1 0
EcoRI 1.0, 3.0, 3.7Kb 3 1 2
PstI 2.0 and 5.7 2 1 1
XbaI 2.7 and 5.0 2 Insertion site 0
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Map of PstI 2 and 5.7Kb
5.0
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Map of EcoRI 1, 3 and 3.7Kb