PROBER Tutorial


Section 5

PROBER: Output the primer and probe sequence files

After probe selection is complete a similar layout of the probes will appear in the main window



The probe layout shows that PROBER found 16 Probes from 'Tier 1' of probe selection and 20 Probes in 'Tier 2'. The distribution of the probes across the genomic region can be visualized along with the total coverage in dark blue at the bottom of the screen. The light blue indicates the total genomic region that was considered for probe design (in our case a 100kb region). Above each rectangle (probe) is the probe identifier (Probe ID) and below is the probe length.

The Percent Genome Covered (PGC) will read 0.0% until it is calculated.

To calculate the PGC go to Calculations > Percent Genome Coverage

The window will update to 33.11936 % for the PGC, which indicates sufficient coverage of this locus for FISH.
In addition the total base pairs covered will update to 33,114bp and the total base pairs will display 99,983bp.

Note : In general, if the PGC is < 20% or less then 20 probes are identified, we recommend using more relaxed parameters. To do so increase the range between probe size in both the Tier1 and Tier2 probe lengths.  Increasing the range between the minimum and maximum Tm's will also increase the number of probes the algorithm can identify. Once the new parameters are set, just press the 'Find Probes' button again and repeat this process until enough probes are identified (we recommend finding at least 20 probes for PCR amplification).

Note : If no probes are identified by relaxing the parameters, then return to the MerMatch & Tolerance program and run the MerMatch algorithm again with the "mer.count.cutoff" set higher by (+1).

The user can also use the zoom menu bar to zoom in to 50% or 200% to get a better look at the probe numbers and their position on the locus.

To save an image of the probe layout in *.jpg format, go to File > Save Image File

To save the FULL REPORT go to File > Save *.probes file > Save Full Probe Report

This report contains the probe identifier (Probe ID), the primer sequences, primer lengths, primer locations (relative to the 1st base pair of the genomic sequence), Primer melting temperatures (Tms), the probe length and the probe DNA sequence. Each probe will appear in the file as follows :

::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::
-TIER-1-PROBES:::::::[FULL PROBE REPORT]::::::::::::::::::::::::::::::
::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::
Probe ID 1
PrimerFseq :GCCATTGGCCGCATGCCATGTG
PrimerRseq :TCCAGTTGATCCTACCACAGATGG
PrimerFseq Unmasked :GCCATTGGCCGCATGCCATGTG
PrimerRseq Unmasked :CCATCTGTGGTAGGATCAACTGGA
PrimerF_length :22
PrimerR_length :24
PrimerF_loc :1522
PrimerR_loc :2549
PrimerF_tm :72
PrimerR_tm :72
Probe Length : 1051
Probe Sequence :
GCCATTGGCCGCATGCCATGTGCCACCTGCGGCTTGTGTCTCACCTGTCATCTGGA
CTCAGCACCCAGGCTGCACGTCTGACACCTGAGAGGCGAGAGAGTGGGGCCGGCCT
AGGAGCCAAGGCTGGGGCCTTGCGCTCTGTCCCCAGGATGGTGGCCTTGTTTGTCC
TAAACACACCCAGCACAGGTTCTGGCTTCCTGACATGCTGTGGAGGCAGGGAGGGT
GGGTGGCCACATGTGCTTGAGGGTTTTCACCCTGGCCCTCAGTTGCCTGCTGTGCG
GGTCCCTGGGGCAGCTGCAGGGGCTCATGGACCCATCAGGGTCTCCACAGCTCCCC
TGCAGTGTGTGCACCCCACAATGTCTGCGGCTCTTCTTCCGGCGTGTCGGGCTTTG
ATCACAGCATAGCCACGTCAGTGGCGTGCGCCTCTCGCACAGGCCATTCTGGGTCT
GGTGGTGCCAGGTGCCGTGACACGCCGTGCTGGGCTTGTGCTGCAGCTGGGTGGTG
TGGCCCTCATTCTCATGTTCCAGCTGCTGGGCAGTGCTCTGCCTGTGTGCTGCGCC
TGCAGGCTGCGTGTGCTGCCGTGGATCTCCTGCATCCCTTGACCCCTCCCGCCATC
AGAGGAAAGGCTGCTCCCCGAGGCACCGCTTCCCTGTGCGGCGCTGCAGAGGGGCC
CTCAGTGTGGCACTCCTCGTCAAAGAAAAATAAAGGCTAGAACTGCACCCCGGATC
ACGCGCTTTCTTTGGGGGGAAAGCATCCCATGTAACCCTCATAGCTCCCCGGGGGT
CGCGTGAGGCACAGACCCCAAGGTCCCCGACCTGTCCTTCAGCAGTGGGCTCACGG
GCAGCGGGCATCAGAAAGTGACCTGCCCTTTGCTCCGCCGGTTTGATTCTGGGTGT
GTGGTGGAGCTTTTTGGGACTCAGGTCATGCGGGAACCCCTCCAGCCTGGCCGCAG
GGCTCCCCACTGTACAGTGTGTTGAGGTGCAGCCCAGGGCTCCTTCCTGGGGAACG
GGAGGCCCCGTGGGGATCCTCCAGTTGATCCTACCACAGATGG

Now save the SHORT REPORTs for both the FORWARD PRIMERS and the REVERSE PRIMERS
These text files are ideal for ordering the primers from an oligo synthesis company.

Note : The 'F Primer Short Report' contains the primer sequences for the forward primers
         The 'R Primer Short Report' contains the REVERSE COMPLEMENT sequence for the reverse primers.

First go to File > Save *.probes file > Save F Primers Short Report, then
       go to File > Save *.probes file > Save F Primers Short Report

The short report contains only the primer ID with the Tier and the primer sequence. This is ideal for ordering primer plates or individual primers directly from oligo synthesis companies.

::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::
:::TIER-1 PROBES:::::::::[SHORT FULL F-PRIMER REPORT]:::::::::::::::
::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::
Primer1_F[T1] GCCATTGGCCGCATGCCATGTG
Primer2_F[T1] AGTTGATCCTACCACAGATGG
Primer3_F[T1] GCTTGCAGCAACGAGCTGCC
Primer4_F[T1] GGTGACGGCCGTGGTTGGTC
Primer5_F[T1] ACCTGGCACCTTCGATGTTG
Primer6_F[T1] CTCAGTTCTTACTGCGCCGCG
Primer7_F[T1] CCAGCGCTGCTGTACGTACCC
Primer8_F[T1] GTCTCACGCTCCAGAGCGG
Primer9_F[T1] GCAGGTGCCGTGGAAGCGGTAG
Primer10_F[T1] GCCGCACTCCTGGTACACCTGG

Congratulations, the PROBER tutorial is now complete - you have completed the probe design for your FISH probes and are now ready to determine the copy number of your locus in cells.

We recommend the following PCR protocol for amplifying FISH probes designed with PROBER. You may need to adjust the PCR conditions depending on the parameters that were used for probe selection. After amplification, the probes can be purified using Qiagen purification columns or phenol/chloroform, however we highly recommend the former.