DNA Sequencing 8.2 Image from: http://www.sciencedaily.com/releases/2009/10/091014130702.htm

DNA sample Image from: http://blogs.discovermagazine.com/80beats/2009/04/20/dna-sampling-of-innocent-until-proven-guilty-people-is-on-the-rise/#.UL3UexzMJAk

in order to sequence or analyze DNA, more than just a small sample is needed

Polymerase Chain Reaction (PCR) a direct method of making many copies of a DNA sequence each cycle doubles the number of DNA molecules (exponential increase) useful in forensics, medical diagnosis and genetic research because a small amount of DNA can be amplified

Taq polymerase from Thermus aquaticus (bacterium that lives in hot springs) not denatured at the high temperatures needed in PCR works optimally at 72ºC Image from: http://www.neb.ca/emailer/taq/taq.html Great example of importance of biodiversity.

Image from: http://www.flmnh.ufl.edu/cowries/amplify.html

PCR Animations Look at step-by-step animation of PCR process first: http://www.dnalc.org/resources/animations/pcr.html …then this excellent interactive “virtual lab” (one of my favourites): http://learn.genetics.utah.edu/content/labs/pcr/

Gel Electrophoresis Image from: http://mandypozo3a.edublogs.org/2011/12/05/gel-electrophoresis-virtual-lab/

Gel Electrophoresis a method of separating molecules based on size DNA migrates through the gel towards a positive electrode smaller fragments move faster through the gel, causing separation by size DNA fragments can be seen by using a stain such as ethidium bromide

Gel Electrophoresis Image from: http://bio1151.nicerweb.com/Locked/media/ch20/electrophoresis.html

Gel Electrophoresis Another excellent interactive animation: http://learn.genetics.utah.edu/content/labs/gel/ Step-by-step slideshow on gel electrophoresis: http://www.dnalc.org/resources/animations/gelelectrophoresis.html

DNA Sequencing Image of AMY1 gene sequence: http://en.wikipedia.org/wiki/File:AMY1gene.png (amylase gene…one of the most variable in humans)

DNA Sequencing chain termination method (Sanger dideoxy method) developed in the 1970s by Frederick Sanger Human Genome Project used 2 methods of sequencing, both involving the Sanger method

Deoxy- vs. Dideoxy Image from: http://www.mokkka.hu/drupal/en/node/8948

(Image from: http://www. nwfsc. noaa

Image from:http://www. sciencedirect

Sanger method Excellent simple narrated animation: http://smcg.cifn.unam.mx/enp-unam/03-EstructuraDelGenoma/animaciones/secuencia.swf

Whole-Genome Shotgun Image from: http://www.sciencedirect.com/science/article/pii/S0168165603002293

So you’ve sequenced it… …what can you do with the data? structural genomics functional genomics bioinformatics

Interesting findings… can sequence a gene and then use computer to find similar sequences in the same genome or in other genomes have discovered many genes of unknown function unexpected degree of similarity between different organisms (e.g., yeast & humans)

$1000 Genome Initiative currently: about 8 days and $10 000 to sequence the human genome the race is on to be able to sequence the genome faster and for less than $1000 nanopore sequencing Image & data (8 days & $10 000) from: http://www.economist.com/node/18304268

DNA Microarray Image from: http://www.uoguelph.ca/biophysics/central/facilities.htm Reproduced with permission from the Office of Science Education, the National Institutes of Health In this schematic: ﾊGREEN represents Control DNA, where either DNA or cDNA derived from normal tissue is hybridized to the target DNA. ﾊRED represents Sample DNA, where either DNA or cDNA is derived from diseased tissue hybridized to the target DNA.ﾊYELLOW represents a combination of Control and Sample DNA, where both hybridized equally to the target DNA. ﾊBLACK represents areas where neither the Control nor Sample DNA hybridized to the target DNA. ﾊEach spot on an array is associated with a particular gene. Each color in an array represents either healthy (control) or diseased (sample) tissue. Depending on the type of array used, the location and intensity of a color will tell us whether the gene, or mutation, is present in either the control and/or sample DNA. It will also provide an estimate of the expression level of the gene(s) in the sample and control DNA.

DNA Microarray allows scientists to pinpoint the functions of specific genes the expression of thousands of genes in a particular cell can be identified simultaneously

Image from: http://dnadestiny.yolasite.com/resources/chip.jpg

Microarray microarray or gene chip can hold from 10 to 2.1 million DNA samples (probes) could be used to compare a “normal” cell with a cancerous cell isolate mRNA from both cells; synthesize labelled cDNA; denature and place on microarray

Image from: http://en.wikipedia.org/wiki/DNA_microarray

DNA Microarray To read the results: RED shows gene is expressed in experimental (cancerous) cell GREEN show gene is expressed in normal cell YELLOW shows gene is expressed in both cells

Microarray Animation Excellent step-by-step animation with simplified explanation of process: http://www.bio.davidson.edu/courses/genomics/chip/chipQ.html