Alu-PCR hybridization
Pooling strategy
Alu-PCR products (amplified individually with A33 Alu primer,
Chumakov et al, Nature Genetics, 1, 222-225, 1992)
of 24576 YACs (from 734_a_1 to 989_h_12) were pooled according
to a 3-dimensional scheme. This library was divided in 64 superpools of 4
microtiter plates each. Then each superpool (384 clones) was represented by
22 pools:
- 8 pools of half-plates (the first pool contains all half of the first
plate, the second pool contains the second half of the first plate, and
so on). Each of these pools contains Alu-PCR products of 48 YACs.
- 8 pools containing Alu-PCR products of the 64 clones of lanes A to H.
- 6 pools containing columns 1+7, 2+8, 3+9, ... , 6+12 ( Alu-PCR products of
64 clones each).
The total number of pools to test for screening this library is 1408
(64x22). All of them can be contained in a 16-density filter of the size
of a microtiter plate.
Monosomic somatic cell hybrid panel
In order to get information about chromosomal location of each Alu-PCR
probe, we added tin the membranes two copies of a monosomic cell hybrid panel.
This panel comes mainly from NIGMS mapping panel 2. We spotted on membranes
Alu-PCR product of these hybrids.
Membranes construction and hybridization
So far, 4000 membranes have been realised for this project. Each membrane
contains:
- 1408 pools of Alu-PCR products of DNA representing 24576 YACs
- two copies of a monosomic cell hybrid panel allowing chromosomal
assignation of the Alu-PCR probe
- identification and quality control dots
Hybridizations were performed using 33P labeled total Alu PCR product from
individual YACs. About 80% of hybridizations were scored as succesful ,
giving on average 10 related YACs. For 80 % of such cases hybridization signal
to maping panel was also detected.
For the interpretation of the results of both the chromosomal assignation and
related YACs, the following limiting factors should be taken into account:
- the CEPH YAC library contains 30-40% of chimeric clones
- Many regions in the human genome are duplicated
- "Monosomic" somatic cell hybrids often contain pieces from other
chromosomes
The approach described in our Nature publication and realized in
QUICKMAP takes into account these limitations
by computing all interpretations on the fly, the chromosomal assignation
depending on the context. The infoclone program
queries the database without this contextual interpretation, for its goal
is to retrieve exhaustive information about a YAC or an STS.
Image analysis
Autoradiogramms (each of them containing information about 12 membranes) are
scanned with a Truvel scanner. Then a software (dots) developped by the COSE
company calculates coordinates of the dots. Hybridization values (Strong,
Medium, Faint) are computed throuch a program developped at Généthon
(calcule_type). A visualisation program (Xdots), also developped by COSE,
allows manual correction on the films (due to the presence of stains or other
artifacts).
contact: Ilya M. Chumakov (email: chumakov@genethon.fr)
Isabelle Le Gall
Maria Belova, Hung Bui, Pascal Soularue, Jean Luc Sambucy, Gwenael Primas,
Catherine Massard, Catherine Guidicelli
Computer analysis: P. Rigault, E. Poullier, P. Gesnouin