Week 3, Lecture 2 How are Probes Labelled? - PowerPoint PPT Presentation

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Week 3, Lecture 2 How are Probes Labelled?

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Title: Week 3, Lecture 2 How are Probes Labelled?


1
Week 3, Lecture 2 How are Probes Labelled?
  • with some contributions from
  • Brian Baranick, Rudy Gonzales, Sue Robles, Cang
    Thai, Jonnie Burton, Apirada Petchpud
  • Revised 2012, SBS

2
How are probes labeled?
  • Enzymatically
  • by using a polymerase to incorporate nucleotides
    that carry a radioactive isotope of phosphorus or
    sulfur (labeled nucleotides)
  • by using a polymerase to incorporate nucleotides
    that carry a small non-radioactive adduct, such
    as fluorescein or digoxigenin (labeled
    nucleotides)
  • by using a terminal transferase to add labeled
    nucleotides
  • by using a kinase to add a radioactive phosphate
    group to a 5 OH
  • Non-enzymatically
  • by chemically or photochemically covalently
    cross-linking a non-radioactive adduct or an
    enzyme to bases in a strand of nucleic acid

3
Radioactively labeled dNTPs
4
Non-radioactively labeled (d)NTPs
Biotin
Digoxigenin
Fluorescein
5
Probes can be
  • Uniformly labeled (across the entire length)
  • Most common labeling for Southerns, Northerns,
    and colony or plaque hybridizations
  • Methods for uniform labeling
  • Random priming (discontinuous i.e., the probes
    may not be as long as the template)
  • cRNA synthesis (continuous)
  • Cross-linking (continuous)
  • Incorporation of labeled dNTP into PCR product
    (continuous)

6
Probes can be
  • End-labeled
  • (usually not used for Southerns and Northerns)
  • 5 end-labeled
  • Phosphorylation of 5 ribose hydroxyl
  • 3 end-labeled
  • Fill-in of recessed ends
  • Creation and fill-in of recessed ends
  • Extension of 3 end of ss DNA

7
Uniform labeling
8
Random priming (uniform)
Imagine 2 single strands
Hexamers anneal to both strands. So, probes
represent both strands. Label is incorporated all
along the new strand. Note the labeled probes are
not as long as the template and can start
anywhere discontinuous.
Picture 6mers
9
Random priming details
  • Need
  • template fragment
  • E.g., isolated from a recombinant plasmid
  • primer
  • Purchased, synthetic random hexa(6)nucleotides
  • Klenow polymerase
  • Contains the polymerase and 3 to 5 exonuclease
    domains of DNA Polymerase I
  • 4 dNTPs, one of which is labeled
  • Result is multiple fragments of varying lengths
    (200-400 nt) and overlapping sequence
    representing the entire length of the probe
    template fragment

10
Examples of molecules cross-linked to DNA
This crosslink was made between the AP and an
amine on a base by a chemical reaction with
formaldehyde.
Cross-linking is another method of uniform
labeling. This method is continuous. This
picture shows an example of a probe labeled by
crosslinking an enzyme, alkaline phosphatase
(AP), to the DNA strand. (AP) has cross-linked
to an amine group on the DNA using formaldehyde
as a crosslinking molecule.
11
On board
  • How does probe hybridize to target?
  • AP is linked to a base ? steric hindrance
  • Spacing of the AP on the probe is important to
    allow sufficient hybridization

12
Examples of molecules that can be cross-linked to
DNA
? Example of a label linked to a reactive
crosslinking molecule, but not yet crosslinked to
the DNA.
13
Cross-linking (uniform)
  • During a cross-linking reaction, a molecule is
    added non-enzymatically at random positions along
    the length of the probe fragment. The label is
    either
  • a small molecule or
  • an enzyme that creates light or color when
    provided with substrate
  • No new nucleic acid is synthesized
  • Need
  • A probe fragment
  • A label molecule covalently prelinked to a
    reactive molecule
  • reactive molecule is called a crosslinker
  • the reactive molecule is used to crossslink the
    label to the DNA probe
  • chemical or light to supply energy for the
    cross-linking reaction.

14
cRNA synthesis Vector insert for cRNA synthesis
To transcribe with T7 RNA polymerase, open with
an RE downstream of the insert, e.g., Hind III.
Note that the insert is in the Pst I site in this
example.
Insert
15
cRNA transcription Open construct with Hind III
or Sph I ? ds DNA
T7 promoter
Vector
Insert
ds DNA
ss RNAs
SP6 promoter
T7 RNA polymerase
Multiple identical copies of ss cRNA, all of
uniform length
How would you make cRNA complementary to the
other strand of the construct?
16
cRNA synthesis details
  • Results in multiple copies of cRNA with identical
    lengths.
  • You choose which promoter you want to use based
    on which strand you wish to transcribe.
  • Construct must be cut so that polymerase can move
    from the promoter of your choice through the
    insert.

17
cRNA synthesis details
  • Need
  • Insert to be used as template in a suitable
    plasmid.
  • Insert must be flanked by promoter sites for
    polymerases
  • Promoters are usually for T7, T3, or SP6 RNA
    polymerase (phage polymerases).
  • No primer.
  • RNA polymerase that recognizes the promoter of
    choice.
  • T7,T3, and SP6 RNA polymerases can all be bought
    from reagent suppliers. Each different
    polymerase recognizes a distinct promoter
    sequence.
  • 4 NTPs (not dNTPs), one of which is labeled.

18
cRNA details
  • Used as
  • probe for Southerns and Northerns
  • probe for ribonuclease protection assays

19
End-labeling
20
5 end-labeling
3
Phosphatase
Polynucleotide kinase ? 32P-ATP
ADP
21
(No Transcript)
22
5 end-labeling details
  • Used primarily for DNA/protein interaction
    studies (more later)
  • Example gel shifts (possibly more about these
    later)
  • Need
  • Polynucleotide kinase
  • ? (gamma)32/33P-ATP
  • DNA strands with free 5 hydroxyl groups
  • Synthetic oligonucleotide can be ordered without
    5 phosphate
  • DS DNA with blunt or 5 overhang ends that have
    been treated with phosphatase to remove the
    phosphate.

23
3 end-labeling details
  • Used for
  • DNA/protein interaction studies (more later)
  • Oligonucleotide hybridization probe

24
Three methods for 3 end-labeling
  • DS DNA fragment
  • Fill-in of recessed 3 ends
  • Klenow dNTPs, one of which is labeled
  • Exonuclease digestion and fill-in of blunt or 3
    overhanging ends
  • T4 DNA polymerase dNTPs, one of which is labeled
  • Note the fill-in methods give defined ends
  • SS oligonucleotides (or could be a 3 overhang)
  • Template-independent addition of dNTPs to 3 ends
  • Terminal deoxynucleotidyl transferase labeled
    dNTP
  • Note the TdT method gives ends of variable
    length.

25
Fill-in of recessed 3 ends
5
3
Klenow dNTPs (one or more labeled)
Klenow is a fragment of DNA polymerase I and
lacks the 5 to 3 exonuclease activity present
in the DNA Pol I holoenzyme.
26
T4 polymerase exonuclease digestion and fill-in
of blunt or 3 overhanging ends
5
3
Note 3 now recessed
3
Try drawing the reaction starting with blunt ends.
27
Template-independent addition of labeled dNTPs to
3 ends of ss DNAs or 3 overhangs
Terminal deoxynucleotidyl transferase (TdT)
dNTPs
Try drawing the reaction starting with dsDNA with
3 overhangs.
28
If you need defined ends, and identical probe
molecules, use
  • cRNA
  • 5 end-labeling
  • 3 end-labeling with T4 or Klenow
  • Cross-linking might work (depends on whether
    reagents break some phosphodiester bonds as a
    side reaction I dont know)
  • (Why wont terminal transferase work for this
    purpose?)

29
Clarification
  • Incorporating label into a new strand of DNA as
    it is being synthesized by extension of primer on
    a template
  • Which methods do this?
  • Adding label on to a pre-existing strand of DNA
  • Which methods do this?

30
RIBONUCLEASE PROTECTION ASSAY
aka-
RIB0NUCLEASE DIGESTION ASSAY WHAT KIND OF
PROBE? Hybridization and nuclease digestion are
in solution. Products of the digestion are run
out on a gel. The gel is subjected to
visualization by autoradiography.
Why is it important to have all probe molecules
of uniform length? Which methods could be used
to label a probe used for this purpose? Why
would an end-labeled probe not be appropriate for
this particular application of ribonuclease
protection?
http//www.gene-quantification.de/mrna-fig-2.gif
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