2 ANÁLISIS ESTRUCTURAL
2.7 DISEÑO DE LA ESTACIÓN DE SALIDA
2.7.3 CARGA DE SISMO PARA LA ESTACIÓN DE SALIDA
2.1.2.1. Amplification of RH panels
To construct radiation hybrid (RH) maps of the linkage regions analysed in this study,
each marker to be mapped was amplified by PCR across the Stanford 0 3 (0 3 ) RH panel
and the MIT/Whitehead Oenebridge 4 (0 4 ) RH panel. The 0 3 and 0 4 RH panels are
comprised of 83 and 93 human-hamster hybrid, respectively. Both panels also include
both a human and hamster positive control. Both panels are supplied with 5pg of DNA
a). OAG.SIBPAIR, 103,3,2,1,2„„SP01 Al 1 ,OAG-IBD-001023
b). PopID,KinTD,SubTD,IBD,CD,UC
OAG.SIBPAIR,103,3,2,2,1
c). D3S 1573,homo,sapiens,3,AFM 126xg7
d). 38,B,SP01 A 11 ,D3S 1573,124,126„RAM Disk;ibd6 rl :ibd6 rl
e). D3S1573 2.3
Figure 2.3. Examples of Discovery Manager^^ data import formats
a). Pedigree data is written in comma delimited fonnat without a header. The data is
divided into the following fields: population identification, kindred identification,
subject identification, mother identification, father identification, sex, date of birth, date
of death, proband, alias 1, alias 2, and alias n^\ The first five fields are required. Sex is
a coded variable where 0 is unknown, 1 is male and 2 is female.
b). Phenotype data is written in comma delimited format with a required header. The
data is divided into the following fields: population identification, kindred
identification, subject identification, phenotype 1, phenotype 2, and phenotype n"\
Phenotypes used in this study were coded variables where 0 was unknown, 1 was
unaffected and 2 was affected.
c). Marker data is written in comma delimited format without a header. The data is
divided into the following fields: marker name, genus, species, chromosome, alias 1,
alias 2, and alias n*.
d). Genotype data is written in comma delimited format without a header. The data is
divided into the following fields: lane, dye, subject alias, marker, allele 1, allele 2,
overflow, and gelfile.
e). Map data is written in tab delimited format without a header. The data is divided
panel was transferred to a deep 96-well plate in the order specified for submission of
results, starting with wells Al - A12 and then B1 - B12, etc. For both panels, the
human control was placed in well A4 and the hamster control in A8. Amplification of
each marker was performed using PCR (Section 2.1.1.7.) and 25ng of each DNA (5pi)
from each panel. The amplification of each marker was performed in duplicate to verify
results.
2.1.2.2. Visualisation of RH panel amplification
After amplification of a marker across one of the RH panels, the resulting reactions
were fractionated and stained with ethidium bromide by agarose gel electrophoresis
(Section 2.1.1.8.). The agarose gels were constructed using 52-well combs. When
loading the PCR reactions onto the agarose gel each specific DNA amplification was
loaded next to its duplicate reaction. Thus, for each maker and RH panel four lanes of
52 wells were used. Lane one contained PCR reactions from row A and B of a 96-well
plate, loaded: lOObp ladder, A l, Al duplicate, A2, A2 duplicate, etc. Lanes two, three,
and four contained rows C and D, E and F, and G and H, respectively. After
electrophoresis the DNA fragments were visualised, imaged and stored (Section
2.I.I.9.).
2.1.2.3. Analysis of RH data and map construction
Each image of an amplification of a marker across an RH panel was analysed for
validity and then scored. To validate the RH mapping data, the negative controls, wells
H2 - H I2 for the 0 3 panel and H I2 for the 0 4 panel, and positive controls, wells A4
and A8 for both panels, were analysed. The data was considered valid if the negative
controls were blank, the human controls had one DNA band present of the correct size
contained a band(s) of a different size to the positive control which would not interfere
with the calling of data from human-hamster hybrid PCR. The remainder of the
duplicate data was scored in the sequence of clones specified for each RH panel. For
each clone, two positive results (the presence of a band the same size as the human
control band) were scored as a 1, two negative results (no band matching the human
control band) were scored as a 0, and one positive and one negative result were scored
as a 2 for the G4 panel or as a R for the G3 panel. The data was used to place the
marker on a pre-existing framework RH map and to construct new RH maps solely from
RH data generated from this project.
2.1.2.3.1. Stanford RHServer
Data generated from the amplification of a marker across the G3 RH panel was
submitted through the Stanford RHServer Internet site for analysis. The chromosomal
assignment expected was selected. The closest five markers with a resulting LOD score
of greater than 4.0 were designated for retrieval. After submission, results were
returned by email (Figure 2.4.). By comparing the resulting distances between the
marker submitted and the markers retrieved to the order of the retrieved markers on the
G3 RH map, the new marker could be placed on the map (see example - Figure 2.4.).
After all markers from a particular chromosome were placed on the map, the distances
between each submitted marker were calculated using the distances given on the G3 RH
map and a G3 RH map consisting of only the submitted markers was constructed.
2.1.2.3.2. MTT/Whitehead Institute
Data generated from the amplification of a marker across the G4 RH panel was
submitted to the Whitehead Institute/MIT Center for Genome Research RH server
a).
G3 .D3S1573 00000001100010000000000000000010000000000000000100100000
OOOOOOOROOO1RO1000001010111
b).
# SHGCNAME CHROM# LOD_SCORE DIST.(cRs)
1 SHGC-1705 3 9.56 13 Vector;0000000110000000000000000000000000110000100000010000000000000011
0010000000001000001
2 SHGC-56838 3 8.71 14 Vector;0000000000000000000000000000000000110000100000010000000000000011 0010000000001000001 3 SHGC-21615 3 8.65 18 Vector:0000000100000000000000000001000000110000100000010000000000000011 0110000000001000001Figure 2.4. 0 3 RH data submission and results via the Stanford RHServer a). An example of the format required for submission of G3 RH data.
b). An example of the results received after submission of G3 RH data (only three of
the five markers retrieved are shown). In the above example, if on the G3 RH map
marker SHGC-1705 is IcR above marker SHGC-56838, then the marker submitted
of greater than 3 designated as the cut off for placing the marker on one of the G4 RH
framework maps. After submission, results were returned by email (Figure 2.5.). The
entire chromosome map was returned with all the submitted markers placed on the map
and distanced between markers calculated (see example Figure 2.5.). After all markers
from a particular chromosome were placed on the map, the distances between each
submitted marker were calculated and a G4 RH map consisting of only the submitted
markers was constructed.
2.1.2.3.3. RHMAP
To construct an RH map without framework markers and derived only from RH data
generated through this project, RHMAP (version 2.01 ) (Boehnke et ciL, 1991) was used. The data for analysis was imported into the program (Figure 2.6.) and analysed using a
multiple point maximum-likelihood test. Possible marker orders were written to a
‘results’ file by descending LOD score. The LOD score associated with each order was
derived from the likelihood of that order calculated from the number of breaks required
to generated the RH data imported. The program could only analyse eight markers at a
time so multiple rounds of analysis with different combinations of markers were
conducted for each region. From the multiple results the most consistent marker order
was chosen and the distances associated with it used for the final map construction.
2.1.2.4. Conversion of RH maps to genetic maps
The RH maps were converted to genetic maps for linkage analysis. The G3 and G4 RH
panels were generated with different amounts of irradiation, 10,000rad and 3,000rad
respectively, and thus have a different factor for conversion from centiRays (cR) to
centiMorgans (cM). For this project, conversion factors of IcRio.ooo = 25kb, IcR^^ooo =
a).
D14S789 10101 01000 10001 11010 01001 10000 10110 00101 01011 01101 00001
10000 01000 00100 00020 10111 00100 00010 000
b).
D 14S789 Chromosome Chr 14
Places I J l c R from D14S872 (lod > 3.0)
Name Dist Type Vector
D14S872 1.7 F 1010101000100011101000001100101011000101010110110100001100000100000100
00010101110010000010000
D14S789 11.6 P >3.0 1010101000100011101001001100001011000101010110110100001100000100000100 00020101110010000010000 D14S288 7.7 F 1010101000101211101000001100001010000101110010110100001100000100000001 00010101110000000010000Figure 2.5. G4 RH data submission and results via the MITAVhitehead RH server a). An example of the format required for submission of G4 RH data.
b). An example of the results received after submission of G4 RH data (only an excerpt
of the entire map is shown). From the excerpt it can be read that, from the data
submitted for D14S787, D14S789 is located 1.7cR below D14S872 and 11.6cR above
I 8 83 1 2 1.2 1.4 1.5 1.8 2.5 2.6 3.5 3.13 (A2,8(1X,A1),T3,I1) 102
1 0 0 0 0 0 0 0 0
2 0 0 0 0 0 0 0 0
3 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 5 0 0 0 0 0 0 0 0 6 0 0 0 0 0 0 0 0 7 0 0 0 0 0 0 0 0 8 1 1 1 1 1 1 1 1 9 1 1 0 0 0 0 0 01 0 0 0 0 0 0 0 0 0
II 0 0 0 0 0 0 0 0
1 2 0 0 0 0 0 0 0 0
72 0 0 0 0 0 0 0 0 73 0 0 0 0 0 0 0 0 74 0 0 0 0 0 1 0 0 75 0 0 0 0 0 0 0 0 76 0 0 0 0 0 0 0 0 77 1 1 1 1 1 1 1 1 78 0 0 0 0 0 0 0 0 79 0 0 0 0 0 0 0 0 80 0 0 0 0 0 0 0 0 81 0 0 0 0 0 0 0 0 82 0 0 0 0 0 0 0 0 83 1 1 0 0 0 0 0 0 8 1 4 1 0 1 1 1.00 1.2 1.4 1.5 1.8 2.5 2.6 3.5 3.13 0 0.00 3.00 3.00 1.2 1.4 1.5 1.8 2.5 2.6 3.5 3.13Figure 2.6. An example of the import format for RHMAP
The first line indicates the program to be used, the number of markers to be analysed,
the number of hybrids assayed, the number of problems to be performed and whether
the species being analysed is haploid or diploid. The second line is a list of the markers
to be analysed. The third line contains the run parameters. The fourth line indicates
how the information is coded 1-positive, 0-negative, and 2-ambigious. The first four
lines are followed by the data (hybrids 13-71 would normally be included) and the