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Week 3, Lecture 2How are Probes Labelled?

with some contributions fromBrian Baranick, Rudy Gonzales, Sue

Robles, Cang Thai, Jonnie Burton, Apirada Petchpud

Revised 2012, SBS

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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

* = 32 or 33P

*

*

35S

Radioactively labeled dNTPs

Non-radioactively labeled (d)NTPs

Biotin

Digoxigenin

Fluorescein

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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)

* ** * * * * *

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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

*

*

or

5’ 3’

5’ 3’

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Uniform labeling

Random priming(uniform)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.

Imagine 2 single strands

Picture 6mers

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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

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Examples of molecules cross-linked to DNA

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.

This crosslink was made between the AP and an amine on a base by a chemical reaction with formaldehyde.

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

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Examples of molecules that can be cross-linked to DNA

Reactive crosslinking molecule

Label

Example of a label linked to a reactive crosslinking molecule, but not yet crosslinked to the DNA.

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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.

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cRNA synthesisVector + insert for cRNA synthesis

To transcribe with T7 RNA polymerase, open with an RE downstream of the insert, e.g., Hind III.

Insert

Note that the insert is in the Pst I site in this example.

cRNA transcriptionOpen construct with Hind III or Sph I ds DNA

Multiple identical copies of ss cRNA, all of uniform length

How would you make cRNA complementary to the other strand of the construct?

InsertT7 promoterVector

T7 RNA polymerase

SP6 promoter

ds DNA

ss RNAs

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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.

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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.

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cRNA details Used as

probe for Southerns and Northerns probe for ribonuclease protection assays

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End-labeling

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5’ end-labeling

PO4-2

Phosphatase

Polynucleotide kinase + 32P-ATP

PO4-2

HO

+ ADP

3’

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32

32P-ATP

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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.

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3’ end-labeling details Used for

DNA/protein interaction studies (more later)

Oligonucleotide hybridization probe

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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.

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Fill-in of recessed 3’ ends

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.

5’ 3’

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T4 polymerase exonuclease digestion and fill-in of blunt or 3’ overhanging ends

T4 DNA polymerase + dNTPs (polymerase fills in end with dNTP)

T4 DNA polymerase + dNTPs (exonuclease chews back to reveal template)

--

5’ 3’

3’Note: 3’ now recessed

Try drawing the reaction starting with blunt ends.

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Template-independent addition of labeled dNTPs to 3’ ends of ss DNAs or 3’ overhangs

Terminal deoxynucleotidyl transferase (TdT)

+ dNTPs

-------

5’ 3’

---------

5’ 3’

Try drawing the reaction starting with dsDNA with 3’ overhangs.

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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 don’t know)

(Why won’t terminal transferase work for this purpose?)

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?

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http://www.gene-quantification.de/mrna-fig-2.gif

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?

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.

RIBONUCLEASE PROTECTION ASSAY

aka-