10/20/20151 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

10/20/20152 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 33 P * * 35 S Radioactively labeled dNTPs

Non-radioactively labeled (d)NTPs Biotin Digoxigenin Fluorescein

10/20/20155 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) * *******

10/20/20156 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’

10/20/20157 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

10/20/20159 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 (~ nt) and overlapping sequence representing the entire length of the probe template fragment

10/20/ 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

10/20/ 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.

10/20/ 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.

10/20/ 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. Insert Note that the insert is in the Pst I site in this example.

cRNA transcription Open 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? Insert T7 promoter Vector T7 RNA polymerase SP6 promoter ds DNA ss RNAs

10/20/ 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.

10/20/ 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.

10/20/ cRNA details Used as probe for Southerns and Northerns probe for ribonuclease protection assays

10/20/ End-labeling

10/20/ ’ end-labeling PO 4 -2 Phosphatase Polynucleotide kinase +  32 P-ATP PO 4 -2 HO + ADP 3’

10/20/  32 P-ATP

10/20/ ’ end-labeling details Used primarily for DNA/protein interaction studies (more later) Example: gel shifts (possibly more about these later) Need Polynucleotide kinase  (gamma) 32/33 P-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.

10/20/ ’ end-labeling details Used for DNA/protein interaction studies (more later) Oligonucleotide hybridization probe

10/20/ 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.

10/20/ 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’

10/20/ 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’ Note: 3’ now recessed Try drawing the reaction starting with blunt ends.

10/20/ Template-independent addition of labeled dNTPs to 3’ ends of ss DNAs or 3’ overhangs Terminal deoxynucleotidyl transferase (TdT) + dNTPs ’3’ ’3’ Try drawing the reaction starting with dsDNA with 3’ overhangs.

10/20/ 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? 10/20/201529

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-