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Enzymes for manipulating DNA

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					Enzymes for manipulating DNA

*** Buffers and solution conditions***

I. DNA polymerases
III. Kinase and alkaline phosphatase
IV. Nucleases
V. Topoisomerase

Course Readings: 19 and 20
Buffers are crucial for activity of enzymes!

Ideal biochemical buffers:

•pKa between 6 and 8
•Chemically inert
•Polar (soluble and not membrane permeable)
•Non-toxic
•Inexpensive
•Salt and temperature indifferent

Tris: pKa is 8.0

Tris(hydroxymethyl)aminomethane (THAM): the free
base for (pH 7.5-8.5)
Tris-HCl: the acidic form (for pH 7-8)
Tris is widely used, but it isn’t perfect:


•Buffering is weak below pH 7.5 and above pH 9.0
•pH must be measured using a special pH meter
electrode
•Toxic to many types of mammalian cell cultures
•Tris solution pH changes with temperature! Drops 0.03
pH units for each degree C increase
•Tris solution pH changes with concentration! Example:
10mM Tris pH 7.9, 100mM Tris pH 8.0


•Below pH 7.5, use a “Good” buffer: HEPES, Tricine,
BES, MOPS, MES
Enzyme “reaction buffers”:
•Buffer: Tris, HEPES, etc.
•Salt: NaCl, KCl, PO4-, etc.--stabilizes protein
structure, facilitates protein-DNA interactions
•Divalent metal ions: Mg2+, Ca2+, Zn2+, etc.--often
required for enzyme activity
•Glycerol: (for storage)--stabilizes protein
structure
•EDTA: chelates (removes) divalent cations--
important especially for storage, if your enzyme is
especially sensitive to metal ion-dependent
proteases
•Beta mercaptoethanol or dithiothreitol: reducing
agents that prevent illegitimate disulfide bond
formation
•Non-specific protein: Bovine serum albumin (BSA)
   DNA polymerases--making
  copies, adding labels, or fixing
               DNA
E. coli DNA polymerase I --the classic DNA
 polymerase
  – Moderately processive polymerase
  – 3'->5' proof-reading exonuclease
  – 5'->3' strand-displacing (nick-
    translating) exonuclease
  – Used mostly for labelling DNA molecules
    by nick translation. For other purposes,
    the Klenow fragment is usually preferred
             DNA polymerases
• Klenow fragment --the C-terminal 70% of E.
  coli DNA polymerase I; originally prepared as a
  proteolytic fragment (discovered by Klenow);
  now cloned
   – Lacks the 5'->3' exonuclease activity
   – Uses include:
      • Labeling DNA termini by filling in the
        cohesive ends generated by certain
        restriction enzymes
      • generation of blunt ends
      • DNA sequencing
A way of making blunt
ended DNA (repair after
mechanical fragmentation)
A way of radiolabeling DNA
              DNA polymerases

• Native T7 DNA polymerase --highly processive,
  with highly active 3'->5' exonuclease
   – Useful for extensive DNA synthesis on long,
     single-stranded (e.g. M13) templates
   – Useful for labeling DNA termini and for
     converting protruding ends to blunt ends
• Modified T7 polymerase (Sequenase) --lack of
  both 3'->5' exonuclease and 5'->3' exonuclease
   – Ideal for sequencing, due to high processivity
   – Efficiently incorporates dNTPs at low
     concentrations, making it ideal for labeling DNA
        DNA polymerases

• Reverse transcriptase
  – RNA-dependent DNA polymerase
  – Essential for making cDNA copies of
    RNA transcripts

  – Cloning intron-less genes
  – Quantitation of RNA
Reverse transcriptase:

The Km for dNTPs is very high (relatively non-
processive)

Makes a DNA copy of RNA or DNA

      -- but --

The self-primed second strand synthesis is inefficient

“Second-strand” cDNA synthesis is usually done with
DNA polymerase and a primer
How RT works
cDNA library
construction
    using
   reverse
transcriptase
                cDNA Library
                Construction Kit
                (Clontech)
Priming reverse transcriptase:
1) General RNA amplification:
   • Oligo(dT)12-18
   • Random sequence oligonucleotides

2) Specific mRNA
   • Single oligonucleotide sequence
      complementary to your mRNA


NOTE: Reverse transcriptase is error-prone
  (about 1/500 bp is mutated)
        Terminal transferase
• template-independent DNA polymerase
• Incorporates dNTPs onto the 3' ends of
  DNA chains

• Useful for adding homopolymeric tails or
  single nucleotides (can be labelled) to the 3'
  ends of DNA strands (make DNA fragments
  more easily clonable)
     T4 polynucleotide kinase

• Transfers gamma phosphate of ATP to
  the 5’ end of polynucleotides

• Useful for preparing DNA fragments for
  ligation (if they lack 5’ phosphates)
• Useful for radiolabelling DNA fragments
  using gamma 32P ATP as a phosphate
  donor
         alkaline phosphatase

• Catalyzes removal of 5’ (and 3’) phosphates
  from polynucleotides
• Useful for treating restricted vector DNA
  sequences prior to ligation reactions,
  prevents religation of vector in the absence
  of insert DNA

• Lack of vector 5’ phosphates may inhibit
  transformation efficiency? Use only when
  absolutely necessary…
                Nucleases

• Exonucleases
  – Remove nucleotides one at a time from a
    DNA molecule

• Endonucleases
  – Break phosphodiester bonds within a DNA
    molecule
  – Include restriction enzymes
            Exonucleases

• Bal 31
  – Double-stranded exonuclease, operates
    in a time-dependent manner
  – Degrades both 5’ and 3’ ends of DNA

  – Useful for generating deletion sets, get
    bigger deletions with longer incubations
             Exonucleases

• Exonuclease III--double-stranded DNA
  – 3’-5’ exonuclease activity
  – 3’ overhangs resistant to activity, can
    use this property to generate “nested”
    deletions from one end of a piece of
    DNA (use S1 nuclease to degrade other
    strand of DNA)
            Exonucleases

• Exonuclease I
  – 3’-5’ exonuclease
  – Works only on single-stranded DNA
  – Useful for removing unextended
    primers from PCR reactions or other
    primer extension reactions
            Endonucleases


• Dnase I
  – Cleaves double-stranded DNA
    randomly (also cleaves single-stranded
    DNA)
  – Mn++: both strands of DNA cut
  – Mg++: single strands nicked
  – Very useful for defining binding sites
    for DNA binding proteins
     DNAse I
   footprinting



Calibrate the
nicking: 1 hit
per DNA
molecule
                    Drosophila heat-shock
  DNAse I       0   factor
footprinting:

    Gel
                      Sites for
 following            interaction of
footprinting          HSF with DNA
  reaction
                              Topoisomerase
Function:
A restriction enzyme and ligase--all in one

altering the “linking number” in coiled, constrained
(supercoiled) DNA--relaxing DNA twisting during
replication

Model for function:
http://mcb.berkeley.edu/labs/berger/structures.html#modeling
Cloning with topoisomerase
            Topoisomerase
•Topoisomerase catalyzed ligation is
EXTREMELY efficient (>85% of resulting
plasmids are recombinant)--excellent for library
constructions

•Can be used to clone blunt ended DNA (PCR
products, restriction digests), T-overhang PCR
products (from Taq polymerase), and directional
clones

•You have to use their plasmid vectors (ie. forget
about using your favorite lab plasmid unless you
know how to covalently attach topoisomerase)
Enzymes for manipulating DNA

*** Buffers and solution conditions***

I. DNA polymerases
III. Kinase and alkaline phosphatase
IV. Nucleases
V. Topoisomerase

Course Readings: 19 and 20
Cutting and pasting DNA
I.   Restriction and modification
     systems
II. Recognition and cleavage of DNA
     by restriction endonucleases
     (REases)
III. Joining (ligating) DNA molecules
IV. Cloning techniques
                   Discovery of
             restriction/modification
EOP = efficiency of
plating (a measure of
phage virulence)


        = bacteriophage




E. coli K has R/M system
E. coli C has no M system
      Cautions for cloning in E.coli

• Strains with methylases (dam or dcm)
produce methylated DNA--difficult to cleave
with certain enzymes, hard to transform some
strains

• Strains with restriction systems intact will
restrict DNA coming from a host lacking
methylases, or from a host with specific types
of methylations

• Best bet is to delete the restriction systems,
but not all cloning strains have this deletion
             Types of endonucleases
• Type I: multisubunit proteins that function as a single protein
  complex, usually contain two R subunits,two M subunits and one S
  subunit

• Type II: recognize specific DNA sequences and cleave at constant
  positions at or close to that sequence to produce 5’-phosphates and
  3’-hydroxyls. Most useful in cloning!!

• Type III: composed of two genes (mod and res) encoding protein
  subunits that function either in DNA recognition and modification
  (Mod) or restriction (Res)
• Type IV: one or two genes encoding proteins that cleave only
  modified DNA, including methylated, hydroxymethylated and
  glucosyl-hydroxymethylated bases
Mode of action of type II REases

           EcoRI


  5´ ... G^A A T T C ... 3´
  3´ ... C T T A A^G ... 5´

                    EcoRI




5´ ... G^ 3’    5’ A A T T C ... 3´
3´ ... C T T A A 5’     3’ ^G ... 5´
     Example recognition sequences for
                 REases
4-cutters:
 AluI           5´ ... AG^CT ... 3´       blunt ends
 MspI           5´ ... C^CGG ... 3´       5’ overhang (2 bp)
6-cutters
 PvuII          5´ ... CAG^CTG ... 3´     blunt ends
 KpnI           5´ ... GGTAC^C ... 3´     3’ overhang (4 bp)
8-cutters
 NotI           5´ ... GC^GGCCGC ... 3´   5’ overhang (4 bp)
Unusual sites
 MwoI           5´ ... GCNNNNN^NNGC ... 3´       3’ overhang
                3´ ... CGNN^NNNNNCG ... 5´       (3 bp)
       How often does REase cut my
                sequence?
1) Known sequence: scan for sites by computer (eg. at
   www.rebase.neb.com)
2) Unknown sequence: hypothetical calculations
  4 cutter: site occurs randomly every 44 (256) base
     pairs
  6 cutter: every 46 (4096) bp
  8 cutter: every 48 (65536) bp
  But sequences are not distributed randomly (table
     3.4)
3) Sequence context effects
  Some sites are preferred over others by enzyme
    The ligation reaction



Biological function
of ligases:
•Lagging strand
DNA synthesis
•genetic
recombination
•DNA repair
Behavior of cohesive ends
      (overhangs)
         Cloning techniques

A) Modify the ends of the DNAs to make
  foreign DNA sequences more ligate-able
B) Directional cloning (generate easily
  cloned PCR fragments)
C) Treat the vector DNA with alkaline
  phosphatase to improve the efficiency of
  ligation of foreign DNA versus vector
  recircularization
  Creating a recombinant DNA molecule



Plasmid vector:
a cloning vehicle

it can replicate
itself in a bacterial
host and contains a
means for selection
(eg. antibiotic
resistance)
Ligation efficiency depends on the DNA
ends in the reaction
Complementary “sticky” ends
  • Ligation is efficient
  • annealing of complementary overhangs brings 5’P
  and 3’OH into close proximity

“Blunt” ends
   • Ligation is inefficient
   • need high concentrations of ligase and DNA
   • molecular crowding reagents (like PEG 8000)
   improve intermolecular ligation, then dilute to
   promote intramolecular ligation

Follow the manufacturer’s instructions…
Cloning foreign DNA by adding linkers




                            (your DNA
                            molecule should
                            not have EcoRI
                            sites in this
                            case)
Cloning foreign DNA by adding adaptors




                            The advantage
                            of this is you do
                            not need to treat
                            the adaptor-
                            modified DNA
                            with restriction
                            enzyme
Terminal transferase to add polynucleotide
   tails to foreign DNA and vector DNA

  Foreign                           Vector
  DNA                               DNA

                             dTTP
Cloning Taq PCR products

•Taq PCR products have a 3’ “A” overhang
•Prepare vector to have a 3’ “T” overhang


                     HphI leaves T overhangs
Directional cloning
         Directional cloning




This guarantees the orientation of your DNA fragment
Easy cloning: PCR products
            Design PCR primers with built in
            restriction sites (check amplified
            sequence for those sites first!)




                                        Ready for
                                        directional
                                        cloning
 Utility of
  alkaline
phosphatas
e in ligation


                Chances of
                getting
                recombinan
                t product
                are
                improved
Cutting and pasting DNA
I.   Restriction and modification
     systems
II. Recognition and cleavage of DNA
     by restriction endonucleases
     (REases)
III. Joining (ligating) DNA molecules
IV. Cloning techniques
Mobilizing DNA: vectors for
propagation in E. coli

•Plasmids
•Bacteriophage
   M13
   Lambda
•Cosmids and BACs
      Plasmids and transformation

I.   Properties of plasmids
II.  Plasmids as cloning vehicles
     (‘vectors’)
III. Ligation and transformation, and
     identification of recombinant
     plasmids

Course Readings: #21 (plasmids) and
    #22 (antibiotic selection)
                Plasmids
• Extrachromosomal, double-stranded, usually
  circular, supercoiled DNA molecules
• Found in many bacterial species
• Replicate and are inherited independently of
  the bacterial chromosome
• Maintain copy number in cell through an origin
  of replication (replicon)
• Usually have genes coding for enzymes that
  provide benefits for the host bacterium, eg.
  antibiotic resistance
         a generic, minimal plasmid


                                  restriction
antibiotic                        site for
resistance                        cloning

                 pBi430/530
               1500 base pairs
             (a manageable size



                              origin of
                              replication
Replicon -- how the plasmid replicates

  • Governs replication of plasmid and number of
    plasmid copies per cell (“copy number”)
  • A replicon includes:
     – origin of replication (ori: a site on the DNA)
     – associated factors
  • > 30 different replicons known, but most
    plasmids used today have pMB1 (or the close
    relative colE1) replicon
        pMB1/colE1
         replication
        mechanism
    1
        Deletion of Rop
        or mutation of
2       RNA II cause
        increases in
        replication and
        copy number
3

4
      Common plasmids and their stats

PLASMID     REPLICON      COPY #

pBR322      pMB1          15-20

pUC         Modified form  500-700
            of pMB1 (RNAII
            mutation)
pACYC       p15A           18-22

pSC101      pSC101        about 5
          Plasmid copy number
• High copy number plasmids
  – Workhorses of molecular cloning
  – Used for almost all routine manipulation of
    small (<15 kb) recombinant DNAs

• Low copy number plasmids
   – For genes that are lethal or unstable in high
     copy number plasmids
   – For constructing Bacterial Artificial
     Chromosomes (BACs) that can propagate
     large (>100 kb) recombinant DNAs
          Plasmid maintenance
• Plasmids contain selectable markers: genes
  carried by the plasmid that confer functions
  required for host survival
• Selection: only those cells with the plasmid
  will survive
   – Allows transformation (a rare event) to be
     feasible
   – A way to keep cells from losing plasmids
     that may otherwise confer a selective
     disadvantage
     Antibiotic resistance genes
• Beta lactamase (bla): breaks down ampicillin
  and carbenicillin (inhibitors of cell wall
  synthesis). Cells carrying this gene are
  often termed ampr

• CAUTION: Over time beta-lactamase is
  secreted into the medium where it breaks
  down the antibiotic and depletes it.
  Eventually this allows the growth of
  ampicillin/ carbenicillin sensitive cells,
  defeating the selection
        Antibiotic resistance genes
• Chloramphenicol acetyl transferase (CAT):
  inactivates chloramphenicol (cm), which
  normally inhibits peptidyl transferase activity of
  the ribosome (no protein synthesis = dead cell)

• Another use for cm:
  – replication of plasmids with pMB1/colE1
    replicons is not inhibited by cm
  – Cm-treated cells stop growing but continue
    making these plasmids, this is a way to
    amplify plasmid copy numbers prior to a
    plasmid prep
      Antibiotic resistance genes
• Tet A (C ) protein: confers resistance to tetracycline (an
  inhibitor of protein synthesis) by pumping this antibiotic
  out of the cell
• Bacterial aminophosphotransferases: confer resistant to
  kanamycins (aminoglycoside antibiotics that inhibit protein
  synthesis) by transferring the gamma phosphate of ATP to
  a 3’ hydroxyl group of the kanamycin
           The ideal plasmid

1. Confers a readily selectable phenotypic
   trait
2. Has single sites for many restriction
   enzymes
3. Low molecular weight
  -- Gives higher copy #, stability, and
      transforming efficiency
  -- Can accept larger pieces of DNA
  -- Easier to handle (less susceptible to
      breakage)
                 pBR322
• The first widely useful cloning vehicle




                              Created using
                              transposition and
                              restriction/ligation
                              reactions
         Utility of pBR322:
         Clone into sites in the
         Tcr gene, which allows
         identification of
         recombinants--these
         will be amp resistant
         but tet sensitive
         (initially plate on
pBR322   ampicillin, then replica
         plate on tetracycline
         plates).


         But: pBR322 has low
         copy number, large
         size, and too few
         options for cloning
         sites
Boldface indicates the restriction site
is present in only one site within the
plasmid
         pUC plasmids
second generation cloning vectors

• Reduced size (about 2000 bp)
• Multiple cloning site (MCS, also called “poly-
  linker”): unique sites for lots of different
  restriction enzymes
• Very high copy number (mutation in RNA II)
• New “blue-white” screening tool for
  recombinants (“alpha complementation” is
  disrupted by foreign DNA in the MCS)
             Alpha complementation
• Plasmid encodes N-terminus               X-gal
of beta galactosidase (alpha
fragment)
• Host strain encodes the C-
terminus of beta galactosidase
(omega fragment)
• Beta galactosidase function is
only seen in the presence of
both the N- and C-terminal
fragments
• Beta gal function can be
monitored by the cleavage of X-
gal which yields a bright blue
product (blue colonies on a        Bright blue
         An alpha complementing plasmid vector

                                                    (MCS)




                       pUC 19




DNA in the MCS interrupts the lacZ gene (no Beta galactosidase)
          Alpha complementation
• Plasmid encodes N-terminus of beta galactosidase
(alpha fragment), with an MCS
• Foreign DNA in the MCS, no alpha fragment
• No alpha fragment, no B-gal
• No B-gal, no blue color (white colonies)

                                    Colony without
                                    foreign DNA in
                                    MCS

pUC19
transformation                      Colony with
plate                               foreign DNA in
    Third generation cloning vectors:
          specialized plasmids
• Vectors containing bacteriophage RNA polymerase
  promoters: for production of a specific RNA (probe
  synthesis, in vitro translation, etc.)
• Low copy number vectors: for cloning of unstable or
  toxic genes
• Vectors designed for expression of specific proteins (for
  further purification and biochemical characterization).
  Proteins may be synthesized with “tags” to assist in
  purification
        Transformation of E.coli
           with plasmid DNA

• E.coli strain: must be antibiotic sensitive, best if it
  lacks restriction-modification systems
• Make cells take up DNA by
   – Chemical competence
   – Electroporation
   – (natural competence--not E.coli though)
         Chemically competent
          cells-basic method

• Grow cells to A600 of 0.4, spin to get cell
  pellet
• Resuspend cells in CaCl2 (100 mM), pellet
  again
• Resuspend in small volume of
  CaCl2/glycerol
• Freeze cells (-80°C) or go straight to
  transformation protocol
      Transformation of chemically
            competent cells
 DNA binds to cells • Mix DNA and competent cells,
                      on ice for 30 min.
DNA uptake by cells • Heat shock (42°C) for 1.5
                      minutes
      Cells recover
                    • Add growth media, 37°C for 1
                      hour
  Selection occurs
                    • Plate on growth medium plus
                      selection (antibiotic) for the
                      plasmid

                      Efficiency ~ 106 - 107
   If cells are good:
                        cells/microgram plasmid DNA
  Ultra competent cells (chemical)
• 5 x 108 transformants/microgram plasmid
• See protocol 23 of Molecular Cloning ch. 1
• Treat with
   – MnCl2
   – CaCl2
   – KCl
   – Hexammine CoCl2
   – Store in DMSO
• (protocol rather difficult, inconsistent)
• These can be bought
  Transformation by electroporation
• > 109 transformants/microgram DNA (ideally)
• Grow cells to A600 of 0.4
• Centrifuge and resuspend in water + 10% glycerol
  (do this 4 times to reduce conductivity)
• Place cells with DNA in electrode-containing cuvette,
  deliver electrical pulse
• If there is arcing (sparks) transformation efficiency
  will be poor (uneven transfer of charge). To avoid
  this make sure the ion concentration is very low (less
  than 10 mM salt)
When cloning a piece of DNA consider:
1) Choice of vector: what kind of plasmid vector to
use (which restriction sites can be used in the
vector)?

2) Ligating DNA to vector: how will the ligation
reaction be set up to facilitate getting what you want?

3) Moving DNA by transformation: what strain of E.
coli will you transform into? Which method for
transformation?

4) Screening for successful ligation products
(recombinant plasmid DNA): how will the recombinant
plasmids be identified?
Setting up a transformation--how
   will the competent cells be
             treated?
1. No plasmid (negative control, nothing
   should grow on this plate)
2. Supercoiled plasmid of a known
   concentration (to determine efficiency of
   competent cells)
3. Vector DNA (dephosphorylated?) ligated
   without insert DNA (background
   transformants)
4. Vector DNA ligated with insert DNA
   (desired products)
  Example outcome of a successful
transformation: chemically competent
                cells

1)   No DNA--No colonies
2)   2 nanograms (10-9 g, 10-3 micrograms)
     supercoiled plasmid DNA--500 colonies
     (efficiency of cells: 2.5 x 105 transformants per
     microgram DNA)
3)   Vector alone--small number of colonies
4)   Vector plus insert--larger number of colonies
     than for #3
        Identifying recombinant
        plasmid-containing cells
• Alpha complementation: most white colonies
  represent presence of insert DNA blocking
  functional beta galactosidase
• Increase in number of transformants in presence
  of insert vs. absence of insert
   – Insert treated with alkaline phosphatase
   – Directional cloning--preventing religation of
     vector
   – Must screen colonies/plasmids for inserts,
     usually by PCR
Confirm clones by sequencing
Mobilizing DNA: vectors for
propagation in E. coli

•Plasmids
•Bacteriophage
   M13
   Lambda
•Cosmids and BACs

				
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