????(Protein Chips)

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• ??????????????????????
• ???????????????,??????
• ??????????????????????
• ????????
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????(Protein Chips)
a microarray-based high-throughput protein assay
method
Chemiluminescence or Fluorescence based detection
methods can be used to visualize bound
antibodies.
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??????

• Diagnostic immunoassay
• that allows the simultaneous high-throughput
  analysis of known autoantigens. In order to
  quantify antibodies in the sera of patients with
  autoimmune diseases, Recombinant antigens and
  control proteins were immobilized on slides with
  reactive aldehyde groups as replicas in serial
  dilutions of the various antigens thereby
  allowing accurate determination of autoantibody
  titer using minimal amounts of serum.
  Miniaturized and highly paralleled immunoassays
  like these will reduce cost by decreasing reagent
  consumption and improve performance by greatly
  increasing the number of assays that can be
  performed with a single serum sample.
• Protein-Protein Interaction.
• DNA- Protein Interaction

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????(Gene chip)

• ????(Gene chip)?????????(Microarray)??????DNA?????
  ??????????????????????????????????????,??????DNA??
  ,??????????,?????????????????????????

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????????
Southern Northern Blot
Dot Blot
Macroarray
Microarray
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Types of DNA Chips
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Comparison of DNA Chip Technologies
Oligo-Chip cDNA-Chip Genomic Chip
8 n or 20 n lt 2,000 n
gt 50,000 n
genomic analysis
sequencing
expression
expression

• Sensitivity of DNA chip based assays is a
  function of
• Probe and target DNA/RNA (Complexity)
• Chip surface (autofluorescence non-spec. bkg)
• Attachment chemistry/methodology (hyb. efficiency
  crosshyb.)
• Hybridization efficiency (lots of factors)
• Detection technology (signal type, efficiency,
  noise)

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Why Genomic Biochips?

• Screen specimens to determine gene copy number
  changes
• Establish correlations between gene copy number
  changes and disease biology
• Determine the interaction of multiple genes on
  the initiation and progression of disease
• Accelerate development of products for genomic
  disease management to guide therapeutic
  intervention
• Combined with expression chips, gives full
  understanding of disease process

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DNA Chip Technology

• Solid support (glass, plastic, metal, silicon)
• Miniaturized array of DNA (genetic material)
• Work on the biochemical principle of DNA/DNA
  hybridization
• Hybridized probes (DNA molecules) are
  fluorescently labeled

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Assay Formats
Genomic
Expression
1. Extract genomic DNA from tissue
1. Extract mRNA from tissue
Normal Sample
2. Produce cDNA by RT Label
2. Label

3. Mix with labeled reference DNA
3. Mix with labeled reference cDNA
4. Hybridize to Chip
4. Hybridize to Chip
5. Wash and Image
5. Wash and Image
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Areas of Biochip Applications

• Academic research of genetic diseases
• Cancer
• Prenatal genetics
• General genetic diseases
• Infectious diseases
• Drug discovery
• Animal farming/veterinary
• Industrial (fermentation, corrosion)
• Environmental

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???? ?????(gene expression pattern)

• Research Use.
• Clinical Diagnostic Use.

Biological Sample
Functional Information
One DiseaseOne Gene Expression Pattern
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Research UseFrom Sequence to function

• ??Ratio ? ( Cy3/Cy5)
• ? 0.5-2.0 ??????????????????????????????
• Clustering

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Research UseFrom Sequence to function
Challenges

• ??????
• ????

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Research Use From Sequence to function

• ??????????(?)???????????????,??????????????????
  ????????????????????????????,?????????,???????????
  ??????????????????

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Research Use From Sequence to function

• ?????????????????????????????
• ???????????????????????????????????????

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Clinical Diagnostic Use
One DiseaseOne Expression Pattern

• Academic research of genetic diseases
• Cancer
• Prenatal genetics
• General genetic diseases
• Infectious diseases
  
• HAV?HBV?HCV..
  
• HIV
  

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Clinical Diagnostic Use

• ?????????????????????????????,????????????????
  ??(???????????)?DNA?RNA????????????(??????)??
  ,???????????

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Prototype AmpliOnc I Biochip
This biochip contains all genomic regions that
have been reported to be amplified in cancers.
AmpliOncTM I Biochip after hybridization color
composite of red, blue and green image



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Oncogene Targets On the AmpliOnc I Biochip
FGR
NMYC
MYCL1
RAF1
HRAS1
EGFR1
KRAS2
FGFR1
REL
PDGFRA
WNT1
CND1
GLI
INT2
MDM2
CDK4
MYB
MET
PIK3CA
MYC
ABL
FGFR2
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9
10
8
12
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3
6
1
5
4
2
JUNB
AR
PDGFB
HER2
AKT2
20q13
YES1
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21
22
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Y
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FES
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AKT1
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14
13
X
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RDA Protocol

• RNA extraction and cDNA preparation from archived
  tissue specimens(tester and driver)
• Generation of amplified cDNA fragments
  (amplicons)
• Subtractive hybridization of amplicons
• Enrichment of cDNA fragments from differentially
  expressed genes

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Microarray ????RDA

• Shotgun subcloning of RDA fragments
• Picking transformed libraries for long-term
  propagation
• Amplification of RDA inserts in 96-well plate
  format for arraying
• Hybridization of cDNA amplicons to microarrays

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References aboutCoupling of RDA
Microarray

• Schena, M. et al. (1995)Science, 270, 467
• Lockhart,et al.(1996).Nature Biotechnology, 14,
  1675
• DeRisi, et al. (1996). Nature Genet., 14, 457.

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Microarray ??????

• SSH
• Differential Display PCR

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????SNPs STRs analysis

• Single Nucleotide Polymorphisms
• Short Tandem Repeats
• Polymerase
• Ligase

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SNPs Typing by Ligase
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SNPs Typing by Polymerase(1)
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SNPs Typing by Polymerase(2)
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STRs Typing(1)
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STRs Typing(2)
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STRs Typing(3)
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???? LCM???????????
LCM Laser Capture Microdissection
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(No Transcript)
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??
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Gene expression profiles of laser-captured
adjacent neuronal subtypes

• Differential Gene Expression
• between Large- and Small-sized
• Dorsal Root Ganglion (DRG) Rat Neurons, Nissl
  stained
• Large DRG Neurons
• myelinated
• fast-conducting
• transmit mechanosensory information
• Small DRG Neurons
• unmyelinated
• slow-conducting
• transmit nociceptive information

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Gene expression profiles of laser-captured
adjacent neuronal subtypes
Dorsal Root Ganglion (DRG) Rat Neurons, Nissl
stained
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cDNA microarray expression patterns of small (S)
and large (L) neurons
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mRNA enriched in large DRG neurons
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mRNA enriched in small DRG neurons
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???????????
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????Development of therapeutic Drugs

• drug target discovery
• evaluation of animal models of human disease
• test for drug efficacy
• test for drug specificity
• test for drug toxicity

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• ????,??????????,??????

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???????? ???????
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????(In Situ Synthesis)
Light directed oligonucleotide synthesis. A
solid support is derivatized with a covalent
linker molecule terminated with a photolabile
protecting group. Light is directed through a
mask to deprotect and activate selected sites,
and protected nucleotides couple to the activated
sites. The process is repeated, activating
different sets of sites and coupling different
bases allowing arbitrary DNA probes to be
constructed at each site.
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???????(off-chip synthesis)

• ?????
• ??????

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?????
Best!
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?????Chipmaker Pin (Telechem??)
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ArrayIt ChipMaker Pins

• Developed in conjunction with Stanford University
• Single dip in sample ? multiple spots
• Fine (EDM) slot in tip of stainless steel pin
• Take-up volume 250 nL (saves sample)
• Spot volume of 100 to 500 pL
• 100 µm to 250 µm spot size (high density arrays)
• Requires 10 to 30 pre-prints to prime pin

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??????nQUAD Technology
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Characteristics of nQUAD Technology

• Wide dispense range
• Low nanoliter to high microliter
• Excellent linearity
• Precise and accurate
• CVs typically less than 10
• Precision less than ? 5
• Non-contact dispense mechanism
• Easier mechanical alignment (384, 1536,)
• On-the-fly printing possible
• Capable of dispensing onto membranes or slides
• Multiple liquid handling modes
• Aspirate/Dispense
• Continuous Reagent Dispensing

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????????????

• ?????????,????96??384??
• ??????250?500nL,??????100?150pL
• ?????100?150um,????????10um
• ???????????
• ?????????????????????
• ???????????????????

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Cartesian - PixSys Series
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?????? - Cartesian Tech.
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PixSys Series ??

• Combines with TWO dispensing technologies
• Contact and Non-contact printing mode
• ChipMaker quill pins and nQUAD dispensers
• Pin array Spot Size 75 to 200um
• nQUAD quantity 10nL
• 10 µm positioning resolution
• 50 slide capacity

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Methods in Microarray Detection

• CCD
• Confocal Scanning Microscopy

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General Scanning Inc.
???Scanner ?????
ScanArray?5000/4000/3000
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SCANARRAY3000/4000/5000??

• ??????????
• ???DNA?????????
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• ?????lt0.5??/u2
• ?????5um???
• ?????????
• ?????????????

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GENERAL SCANNING - ScanArray System
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Summary(1)????

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Summary(2)????????

• Experiments can be performed at a faster rate
• Users can simultaneously screen hundreds to
  thousands of targets(104 - 106) in a single
  experiment
• Analysis of biochips can be automated through
  hardware/software
• Less reagents are needed for experiments
• Low cost per target with biochips

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Summary(3)?????????????

• Photolithography (Affymetrix)
• Drop-on-demand (Cartesian)
• Surface contact (TeleChem, Cartesian)
• Greater density
• Accelerated automation
• Cost reduction