1. DNA Sequencing
8.2
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2. DNA sample
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3. in order to sequence or analyze DNA, more than just a small sample is needed

4. 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

5. Taq polymerase
from Thermus aquaticus (bacterium that lives in hot springs)
not denatured at the high temperatures needed in PCR
works optimally at 72ºC
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Great example of importance of biodiversity.

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

7. PCR Animations Look at step-by-step animation of PCR process first:
…then this excellent interactive “virtual lab” (one of my favourites):

8. Gel Electrophoresis
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9. 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

10. Gel Electrophoresis
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11. Gel Electrophoresis Another excellent interactive animation:
Step-by-step slideshow on gel electrophoresis:

12. DNA Sequencing
Image of AMY1 gene sequence:
(amylase gene…one of the most variable in humans)

13. 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

14. Deoxy- vs. Dideoxy
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15. (Image from: http://www. nwfsc. noaa

16. Image from:http://www. sciencedirect

17. Sanger method Excellent simple narrated animation:

18. Whole-Genome Shotgun
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19. So you’ve sequenced it…
…what can you do with the data?
structural genomics
functional genomics
bioinformatics

20. 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)

21. $1000 Genome Initiative
currently: about 8 days and $ 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:

22. DNA Microarray
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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.

23. 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

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

25. 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

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

27. 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

28. Microarray Animation
Excellent step-by-step animation with simplified explanation of process: