CAP BIOINFORMATICS Su-Shing Chen CISE. 10/5/2005 Su-Shing Chen, CISE 1

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1 CAP BIOINFORMATICS Su-Shing Chen CISE 10/5/2005 Su-Shing Chen, CISE 1

2 Basic BioTech Processes Hybridization PCR Southern blotting (spot or stain) 10/5/2005 Su-Shing Chen, CISE 2

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5 Southern Blotting for Analyzing Genes E. M. Southern (Edinburgh) has developed the Southern blotting method to digest a DNA population by one or several restriction enzymes. The resulting fragments after separation on gels are transferred to filters. A specific radioactive probe is to hybridize the restriction fragments with the DNA or RNA probe. The Southern blots provide a physical map of restriction sites in a gene. 10/5/2005 Su-Shing Chen, CISE 5

6 Detection of specific DNA fragments by gel-transfer hybridization (Southern blotting) 10/5/2005 Su-Shing Chen, CISE 6

7 cdna s and EST s cdna sequences can discover many new protein coding genes Adams et al, EST s (expressed sequence tags) were developed. In 2000, 4.6 million EST s or 62% of sequences in GenBank of 250 organisms (mouse, rat, C. elegans, Drosophila). 10/5/2005 Su-Shing Chen, CISE 7

8 Gene (cdna) Library Gene libraries made by cloning the population of mrna molecules within specialized cells. Useful to isolate gene segments from noncoding intron regions. The information in mrna is copied into cdna (complementary DNA) using the retrovirus enzyme reverse transcriptase generating DNA segments. 10/5/2005 Su-Shing Chen, CISE 8

9 Gene (cdna) Library Reverse transcriptase requires primers, polya tails at the 3 ends of mrna molecules. The synthesis of cdna runs to the 5 end, makes a hairpin loop turn as the primer. The double-stranded cdna is inserted into a plasmid. Then introduce into E. coli and amplified. 10/5/2005 Su-Shing Chen, CISE 9

10 EST Construction Take a cell or tissue of interest. Isolate mrna and reverse transcribe into cdna. Clone cdna into a vector to make a cdna library. Pick individual clones and sequence the 5 and 3 ends of cdna insert. Deposit the EST sequences into dbest. 10/5/2005 Su-Shing Chen, CISE 10

11 Cell or tissue Isolate mrna and Reverse transcribe into cdna dbest Clone cdna into a vector to Make a cdna library Vectors 5 EST Submit To dbest 3 Pick a clone And sequence the 5 and 3 Ends of cdna insert 10/5/2005 Su-Shing Chen, CISE 11

12 EST Properties Approximately 400 bases. EST s are short, only fragments of genes not complete coding sequences. The Washington University Genome Sequencing Center produces over 20,000 EST s per week. 10/5/2005 Su-Shing Chen, CISE 12

13 EST Properties cdna libraries of organs, brain, liver, lung, skeletal muscle, tissues and cells (e.g., cerebral cortex). Some libraries compare different developmental stages, fetal vs. infant, embryonic vs. neonatal. 10/5/2005 Su-Shing Chen, CISE 13

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16 Microarrays Traditionally, one gene and protein and structure were studied. Insufficient to study cell processes and life sciences. Cluster different genes and proteins to study their relationships, functional hierarchies. EST s are expressed in a cell, organ, and state of health. Microarrays speed up EST clustering. 10/5/2005 Su-Shing Chen, CISE 16

17 Functional Gene Networks Large amount of gene expression data Gene network inference Global biochemical models - functions and regulations Boolean and neural networks Functional genomics: Gene expression mappings 10/5/2005 Su-Shing Chen, CISE 17

18 Microarray analysis Microarrays are mrnas Spotted at high density onto glass chips. Expression of thousands of genes over hundreds of cell states is measured. Identifying coregulated genes is not so simple. Many physicists work in this field-- one recent publication: Super-paramagnetic clustering of data, Eytan Domany, Physica A 263, 158 (1999) 10/5/2005 Su-Shing Chen, CISE 18

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20 Microarray Data 10/5/2005 Su-Shing Chen, CISE 20

21 A technology reshaping molecular biology Thousands of genes and their products (i.e., RNA and proteins) in a given living organism function in a complicated and orchestrated way. However, traditional methods in molecular biology generally work on a "one gene in one experiment" basis, which means that the throughput is very limited and the "whole picture" of gene function is hard to obtain. The new technology, DNA microarray, promises to monitor the whole genome on a single chip so that researchers can have a better picture of the interactions among thousands of genes simultaneously. 10/5/2005 Su-Shing Chen, CISE 21

22 Biochip, DNA chip, DNA microarray, and gene array Affymetrix, Inc. owns a registered trademark, GeneChip, which refers to its high density, oligonucleotide-based DNA arrays. In professional journals, popular magazines, and the WWW the term "gene chip(s)" has been used as a general terminology that refers to the microarray technology. Some have suggested genome chip and protein chip. 10/5/2005 Su-Shing Chen, CISE 22

23 Arrays - macroarrays or microarrays An array is an orderly arrangement of samples. It provides a medium for matching known and unknown DNA samples based on base-pairing rules and automating the process of identifying the unknowns. An array experiment can make use of common assay systems such as microplates or standard blotting membranes, and can be created by hand or make use of robotics to deposit the sample. 10/5/2005 Su-Shing Chen, CISE 23

24 Macroarrays and Microarrays Macroarrays contain sample spot sizes of about 300 microns or larger and can be easily imaged by existing gel and blot scanners. The sample spot sizes in microarray are typically less than 200 microns in diameter and these arrays usually contains thousands of spots. Microarrays require specialized robotics and imaging equipment that generally are not commercially available as a complete system. 10/5/2005 Su-Shing Chen, CISE 24

25 Gene Function Discovery DNA microarray, or DNA chips are fabricated by high-speed robotics, generally on glass but sometimes on nylon substrates. Probes with known identity are used to determine complementary binding, thus allowing massively parallel gene expression and gene discovery studies. An experiment with a single DNA chip can provide researchers information on thousands of genes simultaneously. 10/5/2005 Su-Shing Chen, CISE 25

26 Gene Chip Applications There are two major application forms for the DNA microarray technology: 1) Identification of sequence (gene / gene mutation); and 2) Determination of expression level (abundance) of genes. 10/5/2005 Su-Shing Chen, CISE 26

27 DNA microarray technology Format I: probe cdna (500~5,000 bases long) is immobilized to a solid surface such as glass using robot spotting and exposed to a set of targets either separately or in a mixture. This method, "traditionally" called DNA microarray, is widely considered as developed at Stanford University. Format II: an array of oligonucleotide (20~80-mer oligos) or peptide nucleic acid (PNA) probes is synthesized either in situ (on-chip) or by conventional synthesis followed by on-chip immobilization. The array is exposed to labeled sample DNA, hybridized, and the identity/abundance of complementary sequences are determined. Affymetrix, Inc. 10/5/2005 Su-Shing Chen, CISE 27

28 Diversity of Gene Chips Many companies are manufacturing oligonucleotide based chips using alternative in-situ synthesis or depositioning technologies. The microarray (DNA chip) technology is having a significant impact on genomics study. Many fields, including drug discovery and toxicological research, will certainly benefit from the use of DNA microarray technology. 10/5/2005 Su-Shing Chen, CISE 28

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