spotting protocol

Todd Martinsky techmen at hooked.net
Wed Jun 17 18:20:53 EST 1998


ArrayItTM
Micro-Spotting Solution

Advanced polymeric mixture designed to increase the quality of microarray
biochip fabrication by improving the surface properties of the DNA samples
deposited by direct contact DNA micro-spotting technologies.


Product Description

The ArrayItTM Micro-Spotting Solution is an advanced buffer system
containing a patent-pending mixture of ionic and polymeric materials. Use of
our Micro-Spotting Solution will increase the quality of microarray biochips
prepared by all direct surface contact DNA printing technologies.

Users will appreciate the following features:

Supports multiple printing technologies
Improves micro-spotting consistency (<0.01% feature loss)
Increases sample surface tension which reduces feature size
Improves deposition precision allowing easier data analysis
Increases deposition uniformity which improves quantitation
Greater feature uniformity minimizing grid-to-grid variation
Buffer components wash away during processing
Stabilizes DNA samples for prolonged storage
Arrives pre-mixed and sterile, no preparation required
Sufficient to spot 50 million features

Short Protocol (Steps 1-7):

1. Attach covalently a 5’ amino-linker group to DNA samples.

2. Resuspend amino-modified DNA samples in H20 at the desired concentration.

3. Transfer 4.0 µl of each DNA sample into 96-well or 384-well microplates.

4. Add 4.0 µl per well of ArrayItTM Micro-Spotting Solution.

5. Mix the samples by pipetting up and down 10 times.

6. Print the amino-modified DNA samples onto silylated microscope slides.

7. Process the slides for hybridization.



Complete Protocol (Steps 1-7):

Covalently attach a 5’ amino-linker to oligonucleotides or PCR products
either by modification of the oligonucleotides directly during
oligonucleotide synthesis or by enzymatic incorporation of amino-modified
PCR primers into cDNAs during PCR amplification. The 5’ amino-modification
used most successfully with our surface chemistry is the NH2(CH2)6 linker
from Glen Research. The 5’ amino-linker allows selective binding of the
amino-containing DNA to silylated slides through a Schiff’s base reaction
with aldehyde groups on the chip surface. The selectivity of amino-modified
versus natural, unmodified DNA is ~10:1 for cDNAs and ~10,000:1 for
single-stranded 15-mers. DNA molecules of intermediate lengths exhibit
intermediate discrimination ratios. Once bound to the chip surface, the
covalent amino-modification is stable to a wide range of temperatures and
solvents. The 5’end attachment of the DNA to the chip via the amino group
permits steric accessibility of the bound molecules during the hybridization
reaction. The 5’ amino-modification does not appreciably change the
solubility of the DNA (i.e. oligos and cDNAs with amino-linkages have
solubilities comparable to natural, unmodified DNA.
Re-suspend the DNA samples containing a 5’ amino modification in dH20 at the
desired concentration. For PCR products to be used for gene expression
monitoring, a amino-modified DNA concentration of 0.2-1.0 µg/ml is ideal.
For 15-mer oligonucleotides to be used in mutation detection, an
amino-modified DNA concentration of 10-100 pmole/µl is ideal.
Transfer the amino-modified DNA samples into 96-well or 384-well plates.
This is best performed using a multi-channel pipetting device. Purification
of cDNAs with the ArrayItTM PCR Purification Kits will result in a 96-well
or 384-well format for the cDNA samples. If oligonucleotides are synthesized
in a 96-well format, the oligonucleotides may be obtained commercially in a
96-well or 384-well format.
Once the amino-modified DNA samples are transferred to a 96-well or 384-well
format, add 4.0 µl per well of ArrayItTM Micro-Spotting Solution with a
multi-channel pipetting device.
Mix the amino-modified DNA and the Micro-Spotting Solution thoroughly by
pipetting up and down 10 times. The Micro-Spotting Solution contains a
concentrated mixture of ionic and polymeric components and thorough mixing
is required prior to DNA printing. Failure to mix the samples thoroughly at
this step will result in poor microarray quality!
Print the amino-modified DNA samples onto silylated microscope slides by
placing the 96-well or 384-well plates on a suitable microarraying device.
Sample evaporation can be minimized by placing wetted filter discs (e.g.
Whatman) on the underside of the microplate lid and sealing the microplates
with flexible laboratory film (e.g. Parafilm) before and after printing.
Properly sealed plates containing wetted filter discs can be stored for
several weeks at 4°C without detectable loss of volume or DNA stability. For
best results, use the ArrayItTM ChipMakerTM micro-spotting device for
high-density DNA printing.
Following printing, the slides should be left at room temperature for 24 hrs
to permit thorough drying of the DNA onto the surface of the silylated
slides. This can be accomplished by placing the slides in a slide box with
the lid slightly ajar. Direct open air drying of the slides is not
recommended as dust and debris will accumulate on the microarray surface.
Following the 24 hr drying period, the region on the slide containing the
microarray should be marked on the underside to facilitate the downstream
hybridization and detection steps. This can be accomplished by lightly
scoring the underside of the slide with a diamond pencil. Do not score the
DNA side of the slide. Slides should be processed to remove unbound DNA and
the components of the Micro-Spotting Solution. Many protocols have been used
successfully for the slide processing step. One protocol is given below.
Load six printed, dried and scored slides into the ArrayItTM Wash Station.
Transfer the Wash Station and six slides to a 600 ml beaker containing a
stir bar and wash with vigorous agitation with the following solutions.
Twice in 0.2% SDS at 25°C for 5 min each, twice in dH20 at 25°C for 5 min
each, once in dH20 at 95°C for 2 min, cool to 25°C for 5 min, once in sodium
borohybride solution (1.3 g Na2BH4 dissolved in 375 ml phosphate buffered
saline, then add 125 ml pure ethanol) at 25°C for 5 min, three times in 0.2%
SDS for 1 min each, twice in dH20 at 25°C for 1 min each. Air dry the slides
to completion. Slides are ready for hybridization.


Literature Cited

J. Lamture, K.L. Beattie, B.E. Burke, M.D. Eggers, D.J. Ehrlich, R. Fowler,
M.A. Holis, B.B. Kosicki, R.K. Reich, S.R. Smith, R.S. Varma and M.E. Hogan
(1994). Direct detection of nucleic acid hybridization on the surface of a
charge coupled device. Nucl. Acids Res. 22, 2121-2125.
Schena, M., Shalon, D., Heller, R., Chai, A., Brown, P.O., and R.W. Davis
(1996). Parallel Human Genome Analysis: Microarray-Based Expression
Monitoring of 1,000 Genes. PNAS 93, 10614-10619.
Heller, R.A., Schena, M., Chai, A., Shalon, D., Bedilion, T., Gilmore, J.,
Woolley, D.E., and R.W. Davis (1997). Discovery and analysis of inflammatory
disease-related genes using cDNA microarrays. PNAS 94, 2150-2155.


Requirements

5’-Amino-Modifier C6 (Cat.# 10-1906), Glen Research, Sterling, VA

ChipMakerTM Micro-Spotting device (Cat.# CMP-32), TeleChem, San Jose, CA

Wash Station (Cat.# WS-1), TeleChem, San Jose, CA

Silyated Slides (Cat# CCS-100), TeleChem, San Jose, CA



Troubleshooting Tips

Poor printing quality:

Incomplete mixing of DNA samples and Micro-Spotting Solution
Poor DNA attachment:

Forgot to use amino-modified DNA and/or silylated slides
Elevated background fluorescence:

Poor slide processing






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