Literature reviews revised is due4/11 (Friday) turn in together: revised paper (with bibliography) and peer review and 1st draft

available for the test mean variance Chi-square (eq. 15.3)

Mendel1860’s studied “genetics” inheritance and variability Sanger1977 first genome sequenced (phage) genomics -study of entire genome

Human Genome project: Proposed in 1986 Funded in 1989 Preliminary report in 2001 (94%) Completed in 2003 (completed many others too)

© 2006 Jones and Bartlett Publishers Table Some examples of genomic sequencing

© 2006 Jones and Bartlett Publishers Table Some examples of genomic sequencing

Genomics comparative and functional similarities and relationships simple vs. complex gene families gene functions …… Proteomics study the protein content of an organism Normal function Disease processes Repair/drug interaction…..

Genomics Proteomics huge amounts of information computers used to store study compare bioinformatics use of computers to manage and interpret biological information Ivers 2184 pm today

© 2006 Jones and Bartlett Publishers Fig Genes in the genome of Mycoplasma genitalium classified by function. [Data from C. M. Fraser, et al Science 270: 397.]

Genomeresults 1. 95% of human DNA is non-coding (not genes) 2. Fewer genes found than expected (35,000) 3. Many genes have unknown functions 4. Only 1% of our genes are unique (similar to 46% of genes in yeast) genes like bacteria 6. mutation rates differ in different parts of genome 7. Many sites (15) for variability (each individual is genetically unique) 2n2n 8 8,388, = 2 23 =

Even though we know the genetic “information” we are still a long way from understanding how it all functions as an integrated package How would you go about studying?

When do genes get turned “on?” When do genes get turned “off?” What happens when genes are changed? What genes affect other genes? microarrays modifed Southern blot

Fig Southern blot being able to correlate the location of the DNA and its binding to your “probe”

modifed Southern blot DNA-1 DNA-2 DNA-3… DNA sourcescDNA gemonic DNA oligonucleotides being able to correlate the location of the DNA and its binding to your “probe”

microarrays Expression analysis Comparative Genomic Hybridization (CGH) Mutation/Polymorphism analysis look at differential expression look for chromosome rearrangements look for mutations/polymorphisms

microarrays Expression analysis Comparative Genomic Hybridization (CGH) Mutation/Polymorphism analysis bound DNA is cDNA from mRNA pieces of chromosomes oligos (variations on normal genes)

© 2006 Jones and Bartlett Publishers Fig Gene expression microarrays

© 2006 Jones and Bartlett Publishers Fig Small part of a yeast DNA chip. The color of each spot indicates the relative level of gene expression in experimental and control samples. [Courtesy of Dr. Jason Kang/National Cancer Institute]

microarrays

Analysis of information What kind of things can be learned? examples: genetic control of early development protein interactions

Fig 10.15

© 2006 Jones and Bartlett Publishers Fig Two-hybrid analysis by means of a GAL4 protein Fig

© 2006 Jones and Bartlett Publishers Fig Physical interactions among nuclear proteins in yeast. [Adapted from S. Maslov and K. Sneppen, Science 296: 910. © AAAS]

RT-PCR reverse transcriptase PCR mRNA in low #’s Real-Time PCR quantitative q

© 2006 Jones and Bartlett Publishers Real-Time PCR Method of PCR in which the amount of amplicon generated is measured each cycle Quantitative (relative or absolute) Allows comparison of several samples in a single run (experiment) or across multiple runs Uses much less RNA than Northern Blot Amount of amplicon is measured using fluorescence: probe intercalating dye molecular beacon Techniques used in Genomics

© 2006 Jones and Bartlett Publishers Real-Time PCR Probe Method: –5’ and 3’ primers Techniques used in Genomics 5’ 3’ –Probe with reporter dye on 5’ end and quencher on 3’ end –Close proximity of quencher keeps reporter from fluorescing –Taq polyermase adds dNTPs to extend complementary sequence –Taq polymerase 5’ → 3’ nuclease activity cleaves the reporter dye –Reporter dye fluoresces –Each round of PCR will result in more released reporter dye –Thermocycler records amount of fluorescence each round of PCR

© 2006 Jones and Bartlett Publishers Real-Time PCR SYBR Green Method: –5’ and 3’ primers Techniques used in Genomics –SYBR Green intercalates double-stranded DNA and fluoresces –Each round of PCR results in more SYBR Green binding of double-stranded DNA –Thermocycler records amount of fluorescence each round

© 2006 Jones and Bartlett Publishers Real-Time PCR Molecular Beacon Method: Techniques used in Genomics 5’ 3’ –Beacon with reporter dye on 5’ end and quencher on 3’ end –Close proximity of quencher keeps reporter from fluorescing –Taq polyermase adds dNTPs to extend complementary sequence –Beacon hybridizes to complementary sequence –Reporter dye far enough away from quencher to fluoresce –Each round of PCR will result in more hybridized beacon –Thermocycler records amount of fluorescence each round of PCR –In second round of PCR, the copied sequences & beacon are denatured 5’3’