Genetic Engineering

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Genetic Engineering

• technology involved in removing, modifying, or
  adding genes to a DNA molecule
• Aka recombinant DNA technology

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• Restriction endonucleases
• Gel electrophoresis
• Cloning vectors
• Simple cloning exercise

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Nucleases

• exonucleases
• remove single nucleotides from 3'- or 5'-end
  depending on specificity
• most exhibit specificity for either RNA, ssDNA or
  dsDNA
• good for removing undesired nucleic acid or
  removing single stranded overhangs from dsDNA
• endonucleases
• cleaves phoshodiester bonds within fragments
• lack of site specificity limits uses and
  reproducibility

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Restriction Endonucleases
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• Restriction enzymes are classified as
  endonucleases. Their biochemical activity is the
  hydrolysis ("digestion") of the phosphodiester
  backbone at specific sites in a DNA sequence. By
  "specific" we mean that an enzyme will only
  digest a DNA molecule after locating a particular
  sequence.

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• All restriction enzymes cut DNA between the 3
  carbon and the phosphate moiety of the
  phosphodiester bond.

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Origin and function

• Bacterial origin enzymes that cleave foreign
  DNA
• Protect bacteria from bacteriophage infection
• Restricts viral replication
• Bacterium protects its own DNA by methylating
  those specific sequence motifs

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• Named after the organism from which they were
  derived
• EcoRI from Escherichia coli
• BamHI from Bacillus amyloliquefaciens

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Availability

• Over 200 enzymes identified, many available
  commercially from biotechnology companies

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Restriction Enzymes

• site-specific endonucleases of prokaryotes
• function to protect bacteria from phage (virus)
  infection
• corresponding site-specific modifying enzyme
  (eg., methylase)
• type II enzymes are powerful tools in molecular
  biology

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Restriction/modification systems -
EcoRI restriction enzyme
EcoRI methylase
EcoRI
Meth.
EcoRI
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Classes

• Type I
• Cuts the DNA on both strands but at a
  non-specific location at varying distances from
  the particular sequence that is recognized by the
  restriction enzyme
• Therefore random/imprecise cuts
• Not very useful for rDNA applications

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Restriction/modification systems Type III

• R-M systems type III (few examples)- Similar to
  type I- - Recognition sequence 5-7 bp
• - Cleavage site 25-27 bp downstream of
  recognition site (enzyme moves DNA, helicase
  activity)

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• Type II
• Cuts both strands of DNA within the particular
  sequence recognized by the restriction enzyme
• Used widely for molecular biology procedures
• DNA sequence symmetrical
• Reads the same in the 5? 3 direction on both
  strands Palindromic Sequence

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Restriction Enzyme Recognition Sequences

• The substrates for restriction enzymes are
  more-or-less specific sequences of
  double-stranded DNA called recognition sequences.
  
• The length of restriction recognition sites
  varies
• Length of the recognition sequence dictates how
  frequently the enzyme will cut in a random
  sequence of DNA.

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A calculation to ponder

• The enzyme Sau 3A1 cuts on the GATC sequence.
• GATC is something that occurs by chance pretty
  frequently.
• If a DNA sequence is evenly made up of G, A, T,
  and C nucleotides (i.e. 25 of each), we would
  expect to find the sequence GATC" by chance
  about every 256 nucleotides on the average. Why
  is that? Because if we point to a nucleotide in a
  sequence at random, the chances would be one in
  four that it would be G" (the first nucleotide
  in the recognition sequence). The chance that the
  next nucleotide is "A" is also 1 in 4 the chance
  that the nucleotide after that is "T" is 1 in 4
  and the chance that the next one is C" is also 1
  in 4. Therefore, the chance that we have randomly
  pointed to a sequence that reads GATC' is
• (1/4) x (1/4) x (1/4) x (1/4) 1/256

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• Any recognition sequence that was four
  nucleotides in length could be found every 256
  nucleotides (on the average) in this simple
  scenario. In actuality, sequences are usually not
  evenly made up of G, A, T, and C nucleotides,
  which skews the statistics a bit. In addition,
  certain short sequences may be more or less
  common in the DNA, which will also affect the
  frequency with which a recognition sequence is
  found. The dinucleotide CG is very uncommon in
  mammalian DNA, which makes it less likely that
  you will find a recognition sequence for the
  enzyme Hpa II (CCGG).
• Longer recognition sequences lead to lower
  probability of having a site at any point in a
  DNA strand.

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• Enzymes with recognition sequences from 4 to 8
  nucleotides in length each have uses in genetic
  engineering. 6-cutters (i.e. enzymes that have
  recognition sequences specified by six
  nucleotides) are good for day-to-day cloning
  work An example of a 6-cutter is HindIII
  (AAGCTT) which cuts the genome of bacteriophage
  lambda (48 kbp) at 7 sites.

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• 8-cutters are good for carving up chromosomes
  into specific pieces that are still quite large.
  An example of an 8-cutter is NotI (GCGGCCGC) -
  the NotI recognition sequence is not present in
  the genome of bacteriophage lambda.4-cutters
  are good for experiments where you want the
  possibility of cleavage at many potential sites.
  There are 116 Sau3AI sites in the genome of
  bacteriophage lambda.

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Restriction/modification Type II endonucleases
Frequencies of recognition sites 4 bp 44 256
nt 6 bp 46 4096 nt 8 bp 48 65536 nt (NotI
cuts E. coli chromosome 21 times)
Product Blunt end Blunt end 5 overhang 3
overhang Blunt end 5 overhang 5 overhang 5
overhang
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• Restriction recognitions sites can be unambiguous
  or ambiguous The enzyme BamHI recognizes the
  sequence GGATCC and no others - this is what is
  meant by unambiguous. In contrast, Hind II
  recognizes a 6 bp sequence starting with GT,
  ending in AC, and having a Pyrimidine at position
  3 and a Purine at position 4

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• Most restriction enzymes bind to their
  recognition site as dimers (pairs), as depicted
  for the enzyme PvuII in the figure to the right.

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Mechanism of type II restriction endonucleases
Pingoud Jeltsch (2001) Nucl. Acid Res. 29
3705-3727.
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Patterns of DNA Cutting by Restriction Enzymes

• Restriction enzymes hydrolyze the backbone of DNA
  between deoxyribose and phosphate groups. This
  leaves a phosphate group on the 5' ends and a
  hydroxyl on the 3' ends of both strands.

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Types of ends

• 5' overhangs

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• 3' overhangs

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• Blunts

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• Different restriction enzymes can have the same
  recognition site - such enzymes are called
  isoschizomers
• In some cases isoschizomers cut identically
  within their recognition site, but sometimes they
  do not
• Sma I CCC GGG
• Xma I C CCGGG

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Restriction fragments with complementary sticky
ends are ligated easily
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Compatible cohesive ends

• Bam HI G?GATCC
• Bgl II A?GATCT

GTG?GATCCGT CACCTAC?CCA
GTG GATCCGT CACCTAC CCA
CCA GATCTAA GGTCTAG
ATT
CCA?GATCTAA GGTCTAG?ATT
GTGGATCTAA CACCTACATT
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Setting up a digest

• DNA free from contaminants such as phenol or
  ethanol. Excessive salt will also interfere with
  digestion by many enzymes, although some are more
  tolerant of that problem.
• An appropriate buffer Different enzymes cut
  optimally in different buffer systems, due to
  differing preferences for ionic strength and
  major cation. When you purchase an enzyme, the
  company almost invariably sends along the
  matching buffer as a 10X concentrate.
• The restriction enzyme! Remember these are
  generally expensive and heat labile

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Reaction conditions

• 1. A double-stranded DNA sequence containing the
  recognition sequence.2. Suitable conditions for
  digestion.For example, BamHI has the
  recognition sequence GGATCC and requires
  conditions similar to this
• 10 mM Tris-Cl (pH 8.0)5 mM Magnesium
  chloride100 mM NaCl1 mM 2-mercaptoethanolReacti
  on conditions 37 C

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• On the other hand, the enzyme Sma I has the
  recognition sequence CCCGGG and requires
  conditions such as
• 33 mM Tris-acetate (pH 7.9)10 mM Magnesium
  acetate66 mM Potassium acetate0.5 mM
  DithiothreitolReaction conditions 25 C
• Most restriction enzymes are used at 37 C,
  however Sma I is an exception. Other examples of
  temperature exceptions are Apa I (30 C), Bcl I
  (50 C), BstEII (60 C), and Taq I (65 C). Taq I,
  by the way, is a restriction enzyme from the same
  type of organism that produces Taq polymerase
  (Thermophilus aquaticus, or Thermus aquaticus).
  Restriction enzyme names are based on a
  species-of-origin.

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Factors that Influence Restriction Enzyme Activity

• Buffer Composition
• Incubation Temperature
• Influence of DNA Methylation
• Star activity

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• Incubation Temperature The recommended
  incubation temperature for most restriction
  enzymes is 37C. Restriction enzymes isolated
  from thermophilic bacteria require higher
  incubation temperatures ranging from 50C to 65C

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Methylase

• Dam methylase adds a methyl group to the adenine
  in the sequence GATC, yielding a sequence
  symbolized as GmATC.
• Dcm methylase methylates the internal cytosine in
  CC(A/T)GG, generating the sequence CmC(A/T)GG.

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• The practical importance of this phenomenon is
  that a number of restriction endonucleases will
  not cleave methylated DNA.

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• The recognition site for ClaI is ATCGAT, which is
  not a substrate for Dam methylase. However , if
  that sequence is followed by a C or preceeded by
  a G, a Dam recognition site is generated and
  cleavage by ClaI is inhibited. Thus, a random
  sequence of DNA propagated in most strains of E.
  coli, half of the ClaI recognition sites will not
  cut.

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Star Activity

• When DNA is digested with certain restriction
  enzymes under non-standard conditions , cleavage
  can occur at sites different from the normal
  recognition sequence - such aberrant cutting is
  called "star activity". An example of an enzyme
  that can exhibit star activity is EcoRI in this
  case, cleavage can occur within a number of
  sequences that differ from the canonical GAATTC
  by a single base substitutions

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What causes star activity

• High pH (8.0) or low ionic strength (e.g. if you
  forget to add the buffer)
• Glycerol concentrations 5 (enzymes are usually
  sold as concentrates in 50 glycerol)
• Extremely high concentration of enzyme (100 U/ug
  of DNA)
• Presence of organic solvents in the reaction
  (e.g. ethanol, DMSO)

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Unit definition

• The amount of enzyme needed to fully digest 1 ug
  of DNA in 1 hour

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Restriction enzymes cut an organisms DNA into a
reproducible set of restriction fragments
Figure 7-6
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Kpn I
Bam HI
Kpn I
Original plasmid 3500 bp
4k
Bam HI
2k
Digest of original plasmid
1k
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Electrophoresis
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• Electrophoresis is a technique used to separate
  and sometimes purify macromolecules - especially
  proteins and nucleic acids - that differ in size,
  charge or conformation.

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• When charged molecules are placed in an electric
  field, they migrate toward either the positive
  (anode) or negative (cathode) pole according to
  their charge.
• Difference between DNA/RNA and proteins

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• Proteins and nucleic acids are electrophoresed
  within a matrix or "gel".

-agarose or polyacrylamide
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Agarose

• polysaccharide extracted from seaweed. It is
  typically used at concentrations of 0.6 to 2.
  The higher the agarose concentration the
  "stiffer" the gel. Agarose gels are extremely
  easy to prepare you simply mix agarose powder
  with buffer solution, melt it by heating, and
  pour the gel.
• non-toxic.

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Polyacrylamide

• is a cross-linked polymer of acrylamide. 3.5 and
  20.
• Polyacrylamide gels are significantly more
  annoying to prepare than agarose gels. Because
  oxygen inhibits the polymerization process, they
  must be poured between glass plates.
• Acrylamide is a potent neurotoxin and should be
  handled with care

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Uses

• Polyacrylamide gels have a rather small range of
  separation, but very high resolving power.
  polyacrylamide is used for separating fragments
  of less than about 500 bp. However, under
  appropriate conditions, fragments of DNA
  differing in length by a single base pair are
  easily resolved. In contrast to agarose,
  polyacrylamide gels are used extensively for
  separating and characterizing mixtures of
  proteins.
• Agarose is used to separate DNA fragments from
  about 60 bp upward to 100,000 or so bp.

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Visualization of DNA(Agarose)

• Ethidium bromide, a fluorescent dye used for
  staining nucleic acids.
• teratogen and suspected carcinogen and should be
  handled carefully.
• Transilluminator (an ultraviolet light box)

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Gel setup
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• Fragments of linear DNA migrate through agarose
  gels with a mobility that is inversely
  proportional to the log10 of their molecular
  weight. In other words, if you plot the distance
  from the well that DNA fragments have migrated
  against the log10 of either their molecular
  weights or number of base pairs, a roughly
  straight line will appear.

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• Circular forms of DNA migrate in agarose
  distinctly differently from linear DNAs of the
  same mass.

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Circular vs. Linear DNA
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Factors that effect mobility in agarose gels

• Agarose Concentration

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• Electrophoresis Buffer Several different buffers
  have been recommended for electrophoresis of DNA.
  The most commonly used for duplex DNA are TAE
  (Tris-acetate-EDTA) and TBE (Tris-borate-EDTA).
  DNA fragments will migrate at somewhat different
  rates in these two buffers due to differences in
  ionic strength.

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Isolation of DNA from Agarose and Polyacrylamide
Gels

• Electroelution

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• Binding and elution from glass or silica
  particles