K' Further Applications of Microarrays

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K. Further Applications of Microarrays

• K.1. Genome Instability
• K.2. Finding Exon Boundary.
• K.3. Cancer Diagnoistic and Categorization
• K.4. Finding Genomic Changes (Causes) of Cancer
• K.5. Health Care Applications (read Section 5.2
  of GPB)
• K.6. Whats next
• --------------------------------------------------
  -----------------
• Readings Section 4.2. and 5.1 of GPB.

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K.1. Genome Instability Aneuploidy

• Aneuploidy adnormal amount of chromosomal
  material due to duplication (could be partial).
  
• Nulliploid 0 chromosome (some terminally
  differentiated cells red blood cell have no
  nuclei)
• Haploid n chromosome (geme cell) (yeast
  produced asexually)
• Diploid 2n chromosome (almost all mammalian
  cells) (sexually produced yeast).
• Triploid very rare because of problem with cell
  division
• Tetraploid 4n chromosome
• Heart muscle (cardiomyocytes) can be 4n or 8n
• Hepatocytes (80 -- liver) 2n to 8n.
• Aneuploid mutant mutant with extra chromosomal
  material
• Deletion mutant mutant with a gene (typically
  one) deleted (knock out)
• Genome instability sometime part of the
  chromosome is duplicated. Sometime a particular
  chromosome has extra copy(ies). Thought to be
  rare.

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K.1. Genome Instability Revisit Guilt by
Association

• Associate transcription factor to genes that have
  similar expression pattern.
• Sometime, this maybe due to chance.
• Investigators at Rosetta Inpharmatics look at
  yeast microarray notice that 4 genes have nothing
  in common in their promoter region (do not have
  common motif) and do not have similar function.
  Yet, they are co-expressed (with a TF).
• ecm18 - part of cell wall
• erg4 an enzyme to produce lipids

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K.1 Genome Instability Are they really
co-expressed?
wt- Wide-type
Log 1 0
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K.1 Genome Instability Aneuploidy seems to be
more common in the lab.

• Counter to common belief, aneuploidy seem to
  appear in many mutant strains (of yeast) in
  research.
• Suppose a gene, say at chromosome 5, is deleted.
  An isozyme (another gene that may have very
  similar transcript) at another chromosome say 9,
  may take over its function. By having (could be
  partial) aneuploid at that the loci containing
  the isozyme, the mutant may have a better chance
  to survive and hence populate.
• An artifact because they tend to grow faster
  and researchers want to grow yeast

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K.1 Genome Instability Aneuploidy seems to be
more common in the lab.
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K.1 Genome Instability Why labs favor aneuploidy
normal
Deletion mutant
99
1
Deletion mutant
Aneuploidy mutant
Since grow faster, favor by researcher to
culture more mutants
Less favor
95
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Deletion mutant
Aneuploidy mutantv
Amplified by the next generation
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K.1 Genome Instability Deletion of Chromosomal
Material
Repressor gene
Deleted at chromosome 7 As a result, part of
chromosome 13 is lost.
One of the earliest work in microarray
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K.2 Exon Finding and Validation Experimental
Design
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K.2 Exon Finding and Validation Duplicating the
experiment.
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K.2 Exon Finding and Validation Exon Probe
Expression
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K.2 Exon Finding and Validation Validation of
Exons
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K.2 Exon Finding and Validation Refining Exon
Boundary
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K.2 Exon Finding and Validation Over All Exons
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K.2 Exon Finding and Validation Validation
Result
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K.3. Cancer Diagnostic

• Cancer
• Apoptosis programmed cell death
• Cancer do not dead (easily) due to accumulation
  of mutation.
• Movie HHMI Holiday Lecture on Cancer (only need
  to watch the first lecture, although you are
  encouraged to watch the second lecture. The last
  two lectures are more on neuroscience).
• Application in Cancer
• Cancer Diagnostic
• Understanding Genomic Changes in Cancer Cells
• Cancer Treatment

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K.3. Cancer Diagnostic

• DLBCL Diffuse Large B-Cell Lymphoma
• The lymphatic system helps filter out bacteria
  and is important in fighting disease. Every so
  often, the lymph vessels widen into lymph nodes.
• Lymph node Produce B-Cells that in turn
  produces antibody (immunoglobin) that tags on to
  antigens of foreign bacteria or virus, to signal
  foreign object. It signals to the T-cell (white
  blood cell) for destruction.
• B-cell under more mutations than other cell types
  to adapt to the antigens. Sometime they
  hypermutate resulting in cancer.

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K.3. Cancer Diagnostic DCBCL gene expression
analysis

• Figure 5.1 on CD-ROM (too large to fit in here).

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K.3. Cancer Diagnostic Discovery of DCBCL
subtypes
Figure 5.3 on CD-ROM (too large to fit in here).
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K.3. Cancer Diagnostic DCBCL gene expression
signatures
Figure 5.3 on CD-ROM (too large to fit in here).
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K.3. Cancer DiagnoticClinical Distinction of
DLBCL
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K.3. Cancer Diagnotic Understand Treatment
Page 141 143 discuss categorization of breast
cancer. Read it, you are responsible
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K.4. Understanding Genomic Changes in Cancer
Experimental setup

• Instead of determining changes in gene
  expression, want to determine if there is
  aneuploidy. (Similar problem, but not the quite
  the same, to K.1)
• Human genome is too large and too complex to deal
  with.
• Instead, cut the fragments

chromosome
Cut using enzyme at AGATCT
5 . . . A-OH PO4 G A T C T . . .
3 3 . . . T C T A G-PO4 OH-A .
. . 5
Pickup short fragments (lt 1kb)
Amplified using PCR
1658 PCR products
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K.4. Genomic Changes in Cancer Calibration
Individual A for constructing feature
Individual B normal cell to make sure Features
are good.
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K.4. Genomic Changes in Cancer Aneuploidy??
Cancer of B against normal cell of B Note Not
all breast cancer behaves like this. This is for
a particular cell line.
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K.4. Genomic Changes in Cancer Duplicate
experiment
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K.4. Genomic Changes in Cancer Southern Plot
Verification