Introduction to the biology and technology of DNA microarrays

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Introduction to the biology and technology of
DNA microarrays

• Sandrine Dudoit
• PH 296, Section 33
• 10/09/2001

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Biology primer
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The cell

• The basic unit of any living organism.
• It contains a complete copy of the organism's
  genome.
• Humans trillions of cells (metazoa)
• other organisms like yeast one cell
  (protozoa).
• Cells are of many different types (e.g. blood,
  skin, nerve cells, etc.), but all can be traced
  back to one special cell, the fertilized egg.

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The eukaryotic cell
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Eukaryotes vs. prokaryotes

• Prokaryotic cells lack a distinct,
  membrane-bound nucleus.
• E.g. bacteria.
• Eukaryotic cells distinct, membrane-bound
  nucleus.
• Larger and more complex in structure than
  prokariotic cells.
• E.g. mammals, yeast.

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The eukaryotic cell

• Nucleus membrane enclosed structure which
  contains chromosomes, i.e., DNA molecules
  carrying genes essential to cellular function.
• Cytoplasm the material between the nuclear and
  cell membranes includes fluid (cytosol),
  organelles, and various membranes.
• Ribosome small particles composed of RNAs and
  proteins that function in protein synthesis.

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The eukaryotic cell

• Organelles a membrane enclosed structure found
  in the cytoplasm.
• Vesicle small cavity or sac, especially one
  filled with fluid.
• Mitochondrion organelle found in most eukaryotic
  cells in which respiration and energy generation
  occurs.
• Mitochondrial DNA codes for ribosomal RNAs and
  transfer RNAs used in the mitochondrion, and
  contains only 13 recognizable genes that code for
  polypeptides.

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The eukaryotic cell

• Centrioles either of a pair of cylindrical
  bodies, composed of microtubules (spindles).
  Determine cell polarity, used during mitosis and
  meiosis.
• Endoplasmic reticulum network of membranous
  vesicles to which ribosomes are often attached.
• Golgi apparatus network of vesicles functioning
  in the manufacture of proteins.
• Cilia very small hairlike projections found on
  certain types of cells. Can be used for movement.

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Chromosomes
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Chromosomes
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Chromosomes

• The human genome is distributed along 23 pairs of
  chromosomes, 22 autosomal pairs and the sex
  chromosome pair, XX for females and XY for males.
• In each pair, one chromosome is paternally
  inherited, the other maternally inherited.
• Chromosomes are made of compressed and entwined
  DNA.
• A (protein-coding) gene is a segment of
  chromosomal DNA that directs the synthesis of a
  protein.

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Cell divisions

• Mitosis Nuclear division produces two daughter
  diploid nuclei identical to the parent nucleus.
• Meiosis Two successive nuclear divisions
  produces four daughter haploid nuclei, different
  from original cell.
• Leads to the formation of gametes (egg/sperm).

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Mitosis
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Meiosis
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Recombination
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DNA

• A deoxyribonucleic acid or DNA molecule is a
  double-stranded polymer composed of four basic
  molecular units called nucleotides.
• Each nucleotide comprises a phosphate group, a
  deoxyribose sugar, and one of four nitrogen
  bases adenine (A), guanine (G), cytosine (C),
  and thymine (T).
• The two chains are held together by hydrogen
  bonds between nitrogen bases.
• Base-pairing occurs according to the following
  rule G pairs with C, and A pairs with T.

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DNA
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DNA replication
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Genetic and physical maps

• Physical distance number of base pairs (bp).
• Genetic distance expected number of crossovers
  between two loci, per chromatid, per meiosis.
  Measured in Morgans (M) or centiMorgans (cM).
• 1cM 1 million bp (1Mb).

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Genetic and physical maps
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The human genome in numbers

• 23 pairs of chromosomes
• 3,000,000,000 bp
• 35 M
• males 27M, females 44M
• (Broman et al., 1998)
• 30,000-40,000 genes.

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Proteins

• Large molecules composed of one or more chains of
  amino acids.
• Amino acids Class of 20 different organic
  compounds containing a basic amino group
• (-NH2) and an acidic carboxyl group (-COOH).
• The order of the amino acids is determined by the
  base sequence of nucleotides in the gene coding
  for the protein.
• E.g. hormones, enzymes, antibodies.

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Amino acids
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Proteins
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Proteins
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Cell types
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Central dogma

• The expression of the genetic information stored
  in the DNA molecule occurs in two stages
• (i) transcription, during which DNA is
  transcribed into mRNA
• (ii) translation, during which mRNA is translated
  to produce a protein.

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Central dogma
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RNA

• A ribonucleic acid or RNA molecule is a nucleic
  acid similar to DNA, but
• single-stranded
• having a ribose sugar rather than a deoxyribose
  sugar
• and uracil (U) rather than thymine (T) as one of
  the bases.
• RNA plays an important role in protein synthesis
  and other chemical activities of the cell.
• Several classes of RNA molecules, including
  messenger RNA (mRNA), transfer RNA (tRNA),
  ribosomal RNA (rRNA), and other small RNAs.

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The genetic code

• DNA sequence of four different nucleotides.
• Proteins sequence of twenty different amino
  acids.
• The correspondence between DNA's four-letter
  alphabet and a protein's twenty-letter alphabet
  is specified by the genetic code, which relates
  nucleotide triplets or codons to amino acids.

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The genetic code
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Exons and introns
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DNA microarrays
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DNA microarrays

• DNA microarrays rely on the hybridization
  properties of nucleic acids to monitor DNA or RNA
  abundance on a genomic scale in different types
  of cells.

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Nucleic acid hybridization
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Gene expression assays

• The main types of gene expression assays
• Serial analysis of gene expression (SAGE)
• Short oligonucleotide arrays (Affymetrix)
• Long oligonucleotide arrays (Agilent)
• Fibre optic arrays (Illumina)
• cDNA arrays (Brown/Botstein).

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Applications of microarrays

• Measuring transcript abundance (cDNA arrays)
• Genotyping
• Estimating DNA copy number (CGH)
• Determining identity by descent (GMS)
• Measuring mRNA decay rates
• Identifying protein binding sites
• Determining sub-cellular localization of gene
  products

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The process
Building the chip
MASSIVE PCR
PCR PURIFICATION AND PREPARATION
PREPARING SLIDES
PRINTING
RNA preparation
Hybing the chip
POST PROCESSING
CELL CULTURE AND HARVEST
ARRAY HYBRIDIZATION
RNA ISOLATION
cDNA PRODUCTION
DATA ANALYSIS
PROBE LABELING
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(No Transcript)
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(No Transcript)
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The arrayer
Ngai Lab arrayer , UC Berkeley
Print-tip head
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Glass Slide Array of bound cDNA probes 4x4
blocks 16 print-tip groups
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Sample preparation
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Hybridization
cover slip
Hybridize for 5-12 hours
Binding of cDNA target samples to cDNA probes on
the slide
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Hybridization chamber
3XSSC
HYB CHAMBER
ARRAY
LIFTERSLIP
SLIDE
LABEL
SLIDE LABEL

• Humidity
• Temperature
• Formamide
• (Lowers the Tm)

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Scanning
Detector PMT
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RGB overlay of Cy3 and Cy5 images
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Microarray life cyle
Biological Question
Data Analysis Modelling
Sample Preparation
MicroarrayDetection
Taken from Schena Davis
Microarray Reaction
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Biological question Differentially expressed
genes Sample class prediction etc.
Experimental design
Microarray experiment
16-bit TIFF files
Image analysis
(Rfg, Rbg), (Gfg, Gbg)
Normalization
R, G
Estimation
Testing
Clustering
Discrimination
Biological verification and interpretation
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References

• L. Gonick and M. Wheelis. The Cartoon Guide to
  Genetics.
• Griffiths et al. An Introduction to Genetic
  Analysis.
• Access Excellence http//www.accessexcellence.com
  /
• Human Genome Project Education Resources
  http//www.ornl.gov/hgmis/education/education.html
  

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References

• The Chipping Forecast, Nature Genetics, Vol. 21,
  supp. p. 1-60. http//www.nature.com/ng/web_spec
  ials/
• http//stat-www.berkeley.edu/users/sandrine/links.
  html