Targeting Induced Local Lesions In Genomes - or TILLING - was first introduced
in 2000, through the Arabidopsis genome project. The TILLING technique
uses chemical mutagenesis with ethylmethanesulfonate (EMS) to induce point
mutations throughout an entire genome. The mutagenized founder population
is crossbred and the resultant population is analyzed for gene specific
point mutations. EcoTILLING takes regular TILLING one step further in
that it allows for haplotyping groups or species based on natural allele
variation identified through SNP discovery.
Visual Detection of SNPs in a Gel Image
In the TILLING image on the left, the first SNP fragment is detected
in the 700 blue dye channel at position y=1740 pixels. In the image
on the right, the second SNP fragment is detected in the green 800
dye channel at position y=3985 pixels. |
SNPs are detected by comparing the two dye color gel images. The LI-COR
IRDye® 700 – blue-labeled image will show a mutation fragment
band just below the wildtype band. The counterpart band will be visible
in a location exactly opposite of it on the LI-COR IRDye® 800 - green-labeled
image. This fragment in the 800 dye-labeled image is the complementary
cleavage product of the 700 dye-labeled image band. The sum of the length
of these two fragments is equal to the original amplicon, which makes
it easy to distinguish from PCR artifacts. This step can be performed
visually with GelBuddy© software. JelMarker takes it one step further
to detect even the faintest fragments automatically!
Reduction in False Negative SNP Calls
In the Show Mutations window, the blue peak is
at 630bps and the green peak is at 820bps. These SNP fragments were
missed with visual image analysis. |
Converting the gel image to a trace view and performing a physical trace
comparison significantly reduces the amount of time an analyst has to
spend identifying small fragments on a gel image. Converting to traces
also allows for detection of less intense bands that the human eye may
miss resulting in a false negative call. This is especially important
when analyzing EcoTILLING data which contains one mutated portion out
of 12 pooled samples. The SNP fragment in this case would be extremely
faint.
Differential Mobility of Large Fragments is Accounted For
The Edit Size window on the left depicts JelMarker’s
unique size extension algorithm for fragments larger than 700bp.
The image on the right is the Show Mutations window. Notice how
the bp size of the blue and green peaks add up to 1480 basepairs
(with a resolution of approximately 20 basepairs) as expected. |
With JelMarker’s unique Extend Size algorithm, the differential
mobility of large DNA fragments is accounted for. In the example above,
the ladder contains the original basepair sizing from LI-COR’s IRDye
700 Ladder up to size 700bp. From 700bp to 1500bp, the Extend Size feature
in the Edit Size module interpolates the mobility shift of the large peaks
in TILLING data. From those interpolated sizes, an approximate size can
be deduced with a resolution of less than 30 basepairs. Notice the basepair
size ladder at the top of the Mutation trace.
JelMarker was developed in response to a growing demand for software
that can analyze fluorescence, chemiluminescence and autoradiography gel
image files – especially those from LI-COR®’s 4300 DNA
Analyzer, KODAK®’s Image Station 4000R and Hitachi’s FMBIO®
II System. JelMarker can import up to two TIFF, BIP, GEL, JPEG, BMP, IMG
and TXT files for comparison analysis. The software also exports trace
files for easy upload to the fragment analysis software - GeneMarker®.
Application Notes
- TILLING Application Note [PDF]
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