RZPD REsource Center for Genome Research
Im Neuenheimer Feld 506
69120 Heidelberg Germany
telephone: +49 (0)6221 42 4700
fax: +49 (0)6221 42 4704
presenter: Heil, Oliver
O. Heil, M. Roth, A. Poustka and B. Korn
The technology of complex hybridization for expression profiling is dependent on: a) high quality, well characterized RNA material and b) highly specific array elements. These array elements might be oligonucleotides of a given size or melting temperature (oligo arrays) or DNA fragments that are usually amplified by PCR (DNA array). Elements on DNA arrays carry in most cases DNA fragments that represent the complete inserts of given cDNA clones. However, the clone based strategy has many drawbacks, because amplifying inserts by vector primers do always generate products harboring all problematic sequence parts of a given cDNA clone, e.g. polyA tails (length of which is often not even known), repetitive sequences (e.g. Alu), low complex sequences, sequences conserved between different genes (domains, motifs, gene families), and parts of multicloning sites. Furthermore, vector PCR has the intrinsic problem of varying insert sizes, which leads to differences in efficiency in amplification, depending on insert size and GC content of the amplified sequence, resulting in amplification differences of more than 50-fold in molar terms.
In order to circumvent these problems, we have developed algorithms that detect
single copy sequence fragments in all human Genes and EST clusters (also applicable
for other species or whole genomes). In a second step, these sequence parts
can be used for oligonucleotide design: a) for oligo arrays, or b) for PCR primer
design, the approach we currently prefer.
The determination of PCR primers does allow us to choose the sequence part that suits best for PCR. The quality criteria applied are: homogeneous annealing temperature, homogeneous size of PCR product, relative position of the PCR product within each gene, GC-content of PCR product, and potential access for cloning of the individual PCR products.
We have started to amplify all human genes and EST clusters using primer pairs that are designed by this new software tools described above. In total we aim to generate about 70.000 different PCR products within the next 9 months. The algorithms, software tools and data handling will be discussed in detail. In addition, we will present amplification results and discuss automation of data and material handling as well as advantages and disadvantages of this approach towards optimized DNA arrays.
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