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Genomics Analysis Chapter 20. Overview of topics to be discussed. The Human Genome Analysis Variable Number Tandem Repeats Short Tandem Repeats Single Nucleotide Polymorphisms and Haplotypes Expressed sequence tags (ESTs) Predicting Protein Function BLAST searches (in lab)
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Genomics Analysis Chapter 20
Overview of topics to be discussed • The Human Genome Analysis • Variable Number Tandem Repeats • Short Tandem Repeats • Single Nucleotide Polymorphisms and Haplotypes • Expressed sequence tags (ESTs) • Predicting Protein Function • BLAST searches (in lab) • Homlogous genes • Orthologs versus paralogs • Microarray Analysis and an example of its application
Overview Genomic DNA Transcriptome Proteome DATA BASE
The Human Genome Project June 2002
Methods to used include: • The two strategies used: • Bac-to-Bac Sequencing (Map Based Sequencing) • Shot-Gun Method http://www.sciencemag.org/content/291/5507/1304.full
Map-based Cloning of Human Genome “Partial Restriction Digests” Smaller pieces for sequencing
Time Consuming Process whereby: • Areas of overlap between identifies the sequence of DNA http://www.hhmi.org/biointeractive/dna/DNAi_human_genome_seq.html
Shot-Gun Sequencing of the human genome • http://bcs.whfreeman.com/thelifewire/content/chp17/1702002.html
Human Genome approximated: 3.4 billion base pairs 32,000 genes Average gene 27,000 bases Chromosome 17, 19, 22 high density May encode 96,000 proteins
Ongoing data bases from human genome project: • Variable Number Tandem Repeats (VNTRs) • Short Tandem Repeats (STRs) • Single-nucleotide polymorphisms (SNPs) SNP Maps and haplotypes • http://www.ncbi.nlm.nih.gov/pubmed/11029002 • http://www.fruitfly.org/SNP/ • Expressed-sequence tags (ESTs) Oops! Need to understand cDNA
Haplotype:A set of SNPs and other varians on the same chromosome http://hapmap.ncbi.nlm.nih.gov/
Variable number tandem repeats(VNTRs) in multiple loci Short stretches of DNA base pairs that are repeated at multiple loci in the chromosomes. GAG GAGGAG GAGGAGGAG GAGGAGGAG GAGGAGGAGGAG GAGGAGGAG GAGGAGGAGGAGGAGGAG GAGGAGGAGGAGGAG GAGGAGGAGGAG GAGGAGGAGGAGGAGGAG
Forensic Analysis: STRs • FBI: Combined DNA Index System • 13 loci • STRs (4-5 nucleotide repeats)
Example of D1S80 STR • Distance migrated on gel electrophoresis corresponds to size of repeating nucleotide sequence.
cDNA • mRNA • Reverse transcriptase • cDNA
Predicting Function from • Homology Search • Phylogenetic Profile
Computational method prediction • Paralog • Ortholog
Microarray Analysishttp://www.youtube.com/watch?v=VNsThMNjKhM
Microarray Protocol • DNA or cDNA is added robotically to chip • Chips exposed to labeled cDNA • Label monitored by laser
Microarrays from breast tumors • cDNA from breast tumors breast cancer patients EACH patient has a chip and microarray containing “candidate” genes Time passes and some women remain “cancer-free” while other women have recurring cancer. Now return to the original microarrays and compare these two groups of women (disease-free and recurring cancer). Is gene expression different?
The composite microarrayCan you see any differences? (Each row is a patient’s tumor, a column a gene) Cancer free Recurring cancers
Expression patterns varies between patients • Those “genes” that are green from “normal” tissue genes • Those “genes” that are red are associated with cancer!
BLAST searches • Unknowns and examples of how to search database provided tomorrow in lab.
Mouse: Mus musculus • 2.6 billion bp • 99% in common with human genome