Automated sequencing

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Automated sequencing Uses Capillary Array
electrophoresis
96 samples can be analyzed simultaniously
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Also known as BAC by BAC approach
PHYSICAL MAP
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Also known as BAC by BAC approach
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whole genome shotgun sequencing
Advantage less work Disadvantage more gaps in
final assembly
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Positional cloning general strategy
If lt 1 cM apart
average of 12 candidate genes
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Study determinants cell shape Model system
Trichome
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• Screening for trichome morphogenesis mutants
• Two groups
• Branching mutants
• Elongation/Maturation mutants
• We used the two groups to analyze phenotype,
  expression pattern, identification of one of the
  mutants

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Class Two Elongation/Maturation
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Figure from hussey review
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BRK1 experession The When, Where and How
(strong)
Northern Blot
RT-PCR
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Cytoskeleton components
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Actin branching
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ABD2GFP protein fusion
Reports the localization of actin
Wild type
brk1
Differences in actin distribution actin more
bundled in brk1
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Drosophila S2 cells spread on coated glass
surface via actin dependent lamellipodia
RNAi experiment to determine the effects of
Arp2/3 components in animal cells Defects in
actin distribution Defects in adhesion and cell
shape
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Class 2 Branching phenotype
We used only frk from this panel, the others we
did not use
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x
Allelism Tests
How many genes are involved?
M3 plant mut-1/mut-1
M3 plant mut-2/mut-2
Legend
Case 1
Case 2
Mutant phenotype
mut-1/mut-2 Allelism Single gene
mut-1/ mut-2/ No allelism Two genes
Wild-type phenotype
Mutants 1, 2, 3, 5 and 6 were crossed Mutants
1 and 3 mut allelic 3 different genes Mutants 2
and 6 mut allelic
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LINKAGE, CROSSOVER AND MAPPING IN
EUKARYOTES Mapping what does that mean? It
means determining the unknown locus or position
of a gene within the genome relative to other
genes or DNA regions used as markers. T
he Arabidopsis genome has 5 chromosomes as you
know from the TAIR exercise. We want to find the
position of the gene mutated in mutant 1
mut1 ?
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Col
Ler
Variable number of repeats of a certain sequence
(e.g. GA) characterize SSLP markers
Mutation 1 was linked to chromosome 5
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CAPS markers are characterized by the presence of
a restriction site in one allele and the absence
of the restriction site in another allele
Map until you find 0 recombinants with one
molecular marker Mapped mutation one to BAC
K14B20
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Genes on K14B20 are candidates for mutation1 Do
T-DNA lines exist for any of the genes annotated
for BAC K14B20 and if so do they these plants
show a mutant phenotype ?
not all genes of K14B20 are on this presentation
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T-DNA insertion lines
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wild type zwi
At5g65930 SALK_13
wild type
zwi
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Sequencing of ZWI in mutants 1, 2, 3, 5 and 6
Mutant 1 and 3 zwi alleles
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ZWICHEL is a kinesin (motor protein)
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Cytoskeleton components
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Global expansion
Polar expansion
Mathur, 2006
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ZWI ?
BRK1 ARP2/3
Mathur, 2006