Prologue to the science and innovation of DNA microarrays

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Prologue to the science and innovation of DNA microarrays Sandrine Dudoit PH 296, Section 33 10/09/2001

Biology preliminary

The cell The essential unit of any living life form. It contains a total duplicate of the life form's genome . People: trillions of cells (metazoa); different living beings like yeast: one cell (protozoa). Cells are of various sorts (e.g. blood, skin, nerve cells, and so on.), however all can be followed back to one exceptional cell, the treated egg.

The eukaryotic cell

Eukaryotes versus prokaryotes Prokaryotic cells: do not have an unmistakable, film bound core. E.g. microscopic organisms. Eukaryotic cells : particular, film bound core. Bigger and more mind boggling in structure than prokariotic cells. E.g. vertebrates, yeast.

The eukaryotic cell Nucleus: layer encased structure which contains chromosomes, i.e., DNA particles conveying qualities fundamental to cell work. Cytoplasm : the material between the atomic and cell layers; incorporates liquid (cytosol), organelles, and different layers. Ribosome: little particles made out of RNAs and proteins that capacity in protein blend.

The eukaryotic cell Organelles: a film encased structure found in the cytoplasm. Vesicle: little cavity or sac, particularly one loaded with liquid. Mitochondrion: organelle found in most eukaryotic cells in which breath and vitality era happens. Mitochondrial DNA: codes for ribosomal RNAs and move RNAs utilized as a part of the mitochondrion, and contains just 13 conspicuous qualities that code for polypeptides.

The eukaryotic cell Centrioles: both of a couple of tube shaped bodies, made out of microtubules (axles). Decide cell extremity, utilized amid mitosis and meiosis. Endoplasmic reticulum: system of membranous vesicles to which ribosomes are regularly joined. Golgi contraption: system of vesicles working in the make of proteins. Cilia: little hairlike projections found on specific sorts of cells. Can be utilized for development.

Chromosomes

Chromosomes

Chromosomes The human genome is disseminated along 23 sets of chromosomes , 22 autosomal sets and the sex chromosome match, XX for females and XY for guys. In each match, one chromosome is paternally acquired, the other maternally acquired. Chromosomes are made of compacted and weaved DNA . A (protein-coding) quality is a portion of chromosomal DNA that coordinates the union of a protein .

Cell divisions Mitosis: Nuclear division produces two little girl diploid cores indistinguishable to the parent core. Meiosis: Two progressive atomic divisions produces four girl haploid cores, not quite the same as unique cell. Prompts to the arrangement of gametes (egg/sperm).

Mitosis

Meiosis

Recombination

DNA A deoxyribonucleic corrosive or DNA particle is a twofold stranded polymer made out of four essential sub-atomic units called nucleotides . Every nucleotide contains a phosphate gathering, 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-matching happens as per the accompanying tenet: G sets with C , and A sets with T .

DNA

DNA replication

Genetic and physical maps Physical separation: number of base sets (bp). Hereditary separation: expected number of hybrids between two loci, per chromatid, per meiosis. Measured in Morgans (M) or centiMorgans (cM). 1cM ~ 1 million bp (1Mb).

Genetic and physical maps

The human genome in numbers 23 sets of chromosomes; 3,000,000,000 bp; 35 M guys 27M, females 44M (Broman et al., 1998); 30,000-40,000 qualities.

Proteins Large atoms made out of at least one chains of amino acids . Amino acids: Class of 20 distinctive natural mixes containing an essential amino gathering (- NH 2 ) and an acidic carboxyl gathering (- COOH). The request of the amino acids is dictated by the base succession of nucleotides in the quality coding for the protein. E.g. hormones, catalysts, antibodies.

Amino acids

Proteins

Proteins

Cell sorts

Central authoritative opinion The declaration of the hereditary data put away in the DNA particle happens in two phases: (i) interpretation , amid which DNA is deciphered into mRNA; (ii) interpretation , amid which mRNA is meant create a protein.

Central authoritative opinion

RNA A ribonucleic corrosive or RNA particle is a nucleic corrosive like DNA, yet single-stranded; having a ribose sugar as opposed to a deoxyribose sugar; and uracil (U) instead of thymine (T) as one of the bases. RNA assumes an imperative part in protein blend and other concoction exercises of the cell. A few classes of RNA particles, including delivery person RNA (mRNA), exchange RNA (tRNA), ribosomal RNA (rRNA), and other little RNAs.

The hereditary code DNA: arrangement of four distinct nucleotides. Proteins: succession of twenty distinctive amino acids. The correspondence between DNA's four-letter letters in order and a protein's twenty-letter letters in order is determined by the hereditary code , which relates nucleotide triplets or codons to amino acids.

The hereditary code

Exons and introns

DNA microarrays

DNA microarrays DNA microarrays depend on the hybridization properties of nucleic acids to screen DNA or RNA plenitude on a genomic scale in various sorts of cells.

Nucleic corrosive hybridization

Gene expression examines The fundamental sorts of quality expression tests: Serial investigation of quality expression (SAGE); Short oligonucleotide exhibits (Affymetrix); Long oligonucleotide clusters (Agilent); Fiber optic exhibits (Illumina); cDNA exhibits (Brown/Botstein)*.

Applications of microarrays Measuring transcript plenitude (cDNA exhibits); Genotyping; Estimating DNA duplicate number (CGH); Determining personality by drop (GMS); Measuring mRNA rot rates; Identifying protein restricting destinations; Determining sub-cell limitation of quality items; …

The procedure Building the chip: MASSIVE PCR PURIFICATION AND PREPARATION PREPARING SLIDES PRINTING RNA readiness: Hybing the chip : POST PROCESSING CELL CULTURE AND HARVEST ARRAY HYBRIDIZATION RNA ISOLATION cDNA PRODUCTION DATA ANALYSIS PROBE LABELING

The arrayer Ngai Lab arrayer , UC Berkeley Print-tip head

Pins gather cDNA from wells 384 well plate Contains cDNA tests Print-tip aggregate 1 cDNA clones Spotted in copy Print-tip assemble 6 Glass Slide Array of bound cDNA tests 4x4 pieces = 16 print-tip bunches

Sample planning

Hybridization cover slip Hybridize for 5-12 hours Binding of cDNA target tests to cDNA tests on the slide

Hybridization chamber 3XSSC HYB CHAMBER Humidity Temperature Formamide (Lowers the Tm) ARRAY LIFTERSLIP SLIDE LABEL SLIDE LABEL

Image Duplicate spots Scanning Detector PMT

RGB overlay of Cy3 and Cy5 pictures

Microarray life cyle Biological Question Data Analysis & Modeling Sample Preparation MicroarrayDetection Taken from Schena & Davis Microarray Reaction

Biological question Differentially communicated qualities Sample class expectation and so forth. Trial outline Microarray explore 16-bit TIFF records Image examination (Rfg, Rbg) , (Gfg, Gbg) Normalization R , G Estimation Testing Clustering Discrimination Biological check and elucidation

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

References The Chipping Forecast, Nature Genetics, Vol. 21, supp. p. 1-60. http://www.nature.com/ng/web_specials/http://detail - www.berkeley.edu/clients/sandrine/links.html