Plasmid DNA Isolation and Restriction Mapping

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Plasmid DNA Isolation andRestriction Mapping

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• Plasmid DNA isolation
• Agarose gel electrophoresis
• Determine DNA fragment
• size Restriction mapping

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DNA

• Contains all genetic information for that
  organism
• Size is usually expressed in base pairs (bp)
• 2 kinds of DNA
• Chromosomal DNA
• Makes up majority of DNA
• Packed tightly into chromosomes
• Plasmid DNA
• Extrachromosomal DNA
• Small circular molecules, usually 2-10 kb
• Widely used in recombinant DNA technology

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Isolation of DNA

• A good prep should
• Not contain cellular proteins
• Not contain RNA
• Be of high molecular weight

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The alkaline cell lysis method

• Harvest bacterial cells
• Resuspend in Tris-EDTA (RNase, lysozyme)
• Add lysis buffer (NaOHSDS)
• Add sodium acetate PH6.5
• Centrifuge and discard the pellet
• Further purification (special matrix)

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Quality and Quantity of DNA

• Spectrophotometer
• Quantity
• A260 DNA 1 O.D. 50 mg/ml DNA
• ___ O.D. x 50 mg/ml x 100 (dilution) ___
  mg/ml 1 O.D.
• QualityA280 protein Purity A260/A280
• If ratio 2, then assume it is a good prep.
• If ratio gt 2, then there is RNA contamination.
• If ratio lt 1.6, then there is protein
  contamination.

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Quality and Quantity of DNA

• Gel electrophoresis
• Quantity
• Band intensity is semi-quantitative
• Quality
• Look for high MW DNA ? single clear band
• Sheared DNA indicates poor quality ? smear

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Basic Steps in Gene Cloning


Host cell
Transformed host cell
Fragmented genomic DNA or cDNAs from chosen
resource
Recombinant DNA molecules
Vector
Amplification of recombinant molecule
Host cell division
1 gene purified in a clone
Numerous cell divisions ? CLONE
Colonies of transformed host cell clones growing
on solid medium
But how many individual clones needed to
represent the entire genomic DNA or expressed
genes of the resource?
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Techniques of specific cleavage of DNA

• Aim to isolate and manipulate individual genes.
• Means Restriction endonucleases

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What are Restriction endonucleases?

• enzymes that attack and digest internal regions
  of the DNA of an invading bacteriophage but not
  that of the host.
• First enzyme extracted from E. coli (cut randomly
  and not always close to the desired site).

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Types of Restriction endonucleases

• Type I and III Possess both cutting
  (restriction) and protecting activity.
• Protecting activity (modification by
  methylation).
• Type I cuts at random sites.

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Types of Restriction endonucleases

• Type III cuts at specific sites quite near the
  recognition sequence.
• but may be difficult to predict.
• ATP required for source of energy.

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Types of Restriction endonucleases

• Type II restriction enzymes are
• Invariably used in DNA science
• They have several advantages
• 1) has only restriction activity modification
  activity carried by a separate enzyme.

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Types of Restriction endonucleases

• 2) Each cuts in a predictable and consistent
  manner at a site within or adjacent to the
  recognition sequence.
• 3) ATP not needed. Only a cofactor Mg is
  needed.

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Types of Restriction endonucleases

• Type II (continued).
• Today, more than
• 1200 type II have been isolated from a variety of
  prokaryotic organisms
• More than 100 types are commercially available

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Property of restriction enzymes

• They break the phosphodiester bonds that link
  adjacent nucleotides in DNA molecules.

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Nomenclature of Restriction endonucleases

• 1) First letter initial letter of the genus name
  of the organism from which the enzyme is
  isolated.
• 2) Second and third letter usually initial
  letters of the organisms species name.

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Nomenclature of Restriction endonucleases

• 3) Fourth letter (if any) indicates a particular
  strain of organism
• 4) Roman numerals originally indicate the order
  in which enzymes from the same organism and
  strain are eluted from chromatography column.

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Nomenclature of Restriction endonucleases

• More often, though, indicate the order of
  discovery.

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Examples of Type II restriction enzymes

• EcoRI E genus Escherichia
• co species coli
• R strain RY13
• I first endonuclease isolated

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Examples of Type II restriction enzymes

• BamHI B genus Bacilus
• am species
• amyloliquefaciens
• H strain H
• I first endonuclease
• isolated

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Examples of Type II restriction enzymes

• HindIII H genus Haemophilus
• in species influenzae
• d strain Rd
• III third endonuclease isolated

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Type II Restriction Enzymes
Recognize symmetric DNA sequences inverted
repeats
Most are 4-8 base pair long
Cleave within recognition site
5-GAATTC-3 3-CTTAAG-5
Only need Magnesium
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Type II RE practical details
4, 5 or 6 bases
3 types of cuts - 5 overhang, 3 overhang, blunt
This DNA continues on!
5-GAATTC-3 3-CTTAAG-5
5-G-3OH 5P-AATTC-3 3-CTTAA-P5
HO3-G-5
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Type II RE practical details
4, 5 or 6 bases
3 types of cuts - 5 overhang, 3 overhang, blunt
This DNA continues on!
5-GAATTC-3 3-CTTAAG-5
5-GAATT-3OH 5P-C-3 3-C-P5
HO3-TTAAG-5
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Type II RE practical details
4, 5 or 6 bases
3 types of cuts - 5 overhang, 3 overhang, blunt
This DNA continues on!
5-GAATTC-3 3-CTTAAG-5
5-GAA-3OH 5P-TTC-3 3-CTT-P5
HO3-AAG-5
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Agarose Gel Electrophoresis
_

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Sample Well 25 ng 1 kb ladder 0.8 Agarose
Agarose Gel Electrophoresis Sybergold Detection
Limit 0.2-0.5 ng DNA EtBr Detection
Limit 5-10 ng DNA
kb
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10
8
6
4
2
1
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Separation Range Vs. Agarose

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Log (MW) is linearly related to mobility
High has no resolving power at high MW
10 kb
1 kb
Low has no Resolving power at low MW
100 bp
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Star Activity (Relaxation of Specificity)

• . Under certain conditions, the enzymes
  are able to recognize and cleave nucleotide
  sequences which differ in some positions from the
  canonical site.
• For example, under extreme reaction
  conditions (i.e. low ionic strength), BamHI
  (recognition sequence GGATCC)  is able to cleave
  the following sequences NGATCC, GPuATCC and
  GGNTCC. This phenomenon has been called relaxed
  or star activity.

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Star Activity

• In most practical applications of
  restriction endonucleases, star activity is not
  desirable. The analysis of a great number of
  reports on star activity suggest the following
  reasons for this phenomenon
• prolonged incubation time or a large excess of
  enzyme with respect to DNA
• high glycerol concentration (gt5) in the reaction
  mixture or the presence of other organic
  solvents, such as ethanol or dimethyl sulfoxide
• low ionic strength or high pH values in the
  reaction buffer
• substitution of cofactor Mg2 with other divalent
  cation (such as Mn2 or Co2).

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Isoschizomers

• Restriction endonucleases that recognize the same
  sequence are isoschizomers.
• Avi II and Fsp I
• TGC/GCA

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