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Introduction to the biology and technology of DNA microarrays. Sandrine Dudoit PH 296, Section 33 10/09/2001. Biology primer. The cell. The basic unit of any living organism. It contains a complete copy of the organism's genome . Humans: trillions of cells (metazoa);

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Introduction to the biology and technology of DNA microarrays

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Introduction to the biology and technology of dna microarrays l.jpg

Introduction to the biology and technology of DNA microarrays

Sandrine Dudoit

PH 296, Section 33

10/09/2001


Biology primer l.jpg

Biology primer


The cell l.jpg

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.


The eukaryotic cell l.jpg

The eukaryotic cell


Eukaryotes vs prokaryotes l.jpg

Eukaryotes vs. prokaryotes

  • Prokaryotic cells: lack a distinct, membrane-bound nucleus.

    E.g. bacteria.

  • Eukaryoticcells: distinct, membrane-bound nucleus.

    Larger and more complex in structure than prokariotic cells.

    E.g. mammals, yeast.


The eukaryotic cell6 l.jpg

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.


The eukaryotic cell7 l.jpg

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.


The eukaryotic cell8 l.jpg

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.


Chromosomes l.jpg

Chromosomes


Chromosomes10 l.jpg

Chromosomes


Chromosomes11 l.jpg

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.


Cell divisions l.jpg

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).


Mitosis l.jpg

Mitosis


Meiosis l.jpg

Meiosis


Recombination l.jpg

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.


Slide17 l.jpg

DNA


Dna replication l.jpg

DNAreplication


Genetic and physical maps l.jpg

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).


Genetic and physical maps20 l.jpg

Genetic and physical maps


The human genome in numbers l.jpg

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.


Proteins l.jpg

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.


Amino acids l.jpg

Amino acids


Proteins24 l.jpg

Proteins


Proteins25 l.jpg

Proteins


Cell types l.jpg

Celltypes


Central dogma l.jpg

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.


Central dogma28 l.jpg

Central dogma


Slide29 l.jpg

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.


The genetic code l.jpg

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.


The genetic code31 l.jpg

The genetic code


Exons and introns l.jpg

Exons and introns


Dna microarrays l.jpg

DNA microarrays


Dna microarrays34 l.jpg

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.


Nucleic acid hybridization l.jpg

Nucleic acid hybridization


Gene expression assays l.jpg

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)*.


Applications of microarrays l.jpg

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;

  • â€Ļ


The process l.jpg

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


The arrayer l.jpg

The arrayer

Ngai Lab arrayer , UC Berkeley

Print-tip head


Slide42 l.jpg

Pins collect cDNA from wells

384 well plate

Contains cDNA probes

Print-tip group 1

cDNA clones

Spotted in duplicate

Print-tip group 6

Glass Slide

Array of bound cDNA probes

4x4 blocks = 16 print-tip groups


Sample preparation l.jpg

Sample preparation


Hybridization l.jpg

Hybridization

cover

slip

Hybridize for

5-12 hours

Binding of cDNA target samples to cDNA probes on the slide


Hybridization chamber l.jpg

Hybridization chamber

3XSSC

HYB CHAMBER

  • Humidity

  • Temperature

  • Formamide

    (Lowers the Tm)

ARRAY

LIFTERSLIP

SLIDE

LABEL

SLIDE LABEL


Scanning l.jpg

Image

Duplicate spots

Scanning

Detector

PMT


Rgb overlay of cy3 and cy5 images l.jpg

RGB overlay of Cy3 and Cy5 images


Microarray life cyle l.jpg

Microarray life cyle

Biological Question

Data Analysis & Modelling

SamplePreparation

MicroarrayDetection

Taken from Schena & Davis

Microarray Reaction


Slide49 l.jpg

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


References l.jpg

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


References51 l.jpg

References

  • The Chipping Forecast, Nature Genetics, Vol. 21, supp. p. 1-60. http://www.nature.com/ng/web_specials/

  • http://stat-www.berkeley.edu/users/sandrine/links.html


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