DNA Sequencing and Microarrays

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DNA Sequencing and Microarrays

• Janelle Dy, Cameron Kneib, Bailey Robinson

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History of Sequencing

• 1973- Walter Gilbert and Allan Maxam sequence 24
  bp
• 1975- Frederic Sanger develops chain-termination
  method
• 1987- Applied Biosystems sells first sequencing
  machine- ABI 370

http//en.wikipedia.org/wiki/Frederick_Sanger
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Sanger Sequencing and Other Sequencing Methods

• Chain Termination Sequencing- uses radioactive
  markers on primer
• Dye Termination Sequencing- uses florescent ddNTP
  markers
• SMRT Polymerase Sequencing

5gtgtgtgtCTAATTACCATGACAAGAACATTGTATTACTTAAAAACAAGGCA
GTGCTAATGCCTAATGGTGCTACAGTTTCTGCCTCTTCCGTGGAACACAC
ACATGGTGAACTCCTGGAAAAAACACTGTCTCAATATTATCCAGATTGTG
TTTCCATTGCGGTGCAGAAAACCACATCTCACATAAATGCCATTAACAGT
CAGGCTACTAATGAGTTGTCCTGTGAGATCACTCACCCATCGCATACCTC
AGGGCAGATCAATTCCGCACAGACCTCTAACTCTGAGCTGCCTCCAAAGC
CAGCTGCAGTGGTGAGTGAGGCCTGTGATGCTGATGATGCTGATAATGCC
AGTAAACTAGCgtgtgtgt3
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Reagents

• Primer
• dNTPs
• deoxynucleotide
• ddNTPs
• Dideoxynucleotide
• Taq Polymerase
• DMSO
• DNA (PCR product)

http//askabiologist.asu.edu/expstuff/mamajis/sequ
encing/sequencing.html
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Chain Termination
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Sequence determined by Electrophoresis

• Read from bottom to top
• Band indicates a terminated sequence
• However it is often hard to read

http//www.ocf.berkeley.edu/edy/genome/sanger.jpg
http//www.campus.skelleftea.se/biomine/molecular/
images/pic026.gif
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Dye Termination Sequencing

• Can be done in a single tube.
• Accurate
• Fast (high speed capillary electrophoresis)
• Readable

http//www3.appliedbiosystems.com/cms/groups/mcb_m
arketing/documents/generaldocuments/cms_041894.pdf
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• Florescent attached to each ddNTP nucleotide
• Single lane electrophoresis
• Sequence is read by an argon laser that excites
  each florescent bp as it passes by.
• Can only sequence 650800bp per reaction

http//www.bio.davidson.edu/Courses/Molbio/MolStud
ents/spring2003/Obenrader/sanger_method_page.htm
http//en.wikipedia.org/wiki/FileCE_Basic.jpg
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Sequencing Steps
3. Run sequencing reaction

• 1 .Run PCR
• reaction

• 2. Run a gel to qualify PCR product

https//products.appliedbiosystems.com/ab/en/US/ht
docs/productMgr/images/Product-shots-003_thumb.jpg
http//www.mckinneychicago.com/extranet/abiinnovat
ions/02/images/thermalcycler.jpg
http//www.mun.ca/biology/scarr/377_gel_files_narr
ow.jpg
https//sites.google.com/a/luther.edu/genetics/a_/
rsrc/1235684393551/students/eve-doyle/electrophore
sis/Electrophoresis.jpg
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Ethidium bromide Gels
Good Gel
Bad Gel
Can be sequenced
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Background Noise
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Uses

• Human Genome Project
• Forensics
• Paternity Testing
• Identifying diseases and disorders
• Finding similarities between species
• Identifying unknown species in the environment
• Mutations

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What is DNA Microarray Technology?

• Nearly all cells in an organism contain the same
  genes, however each cell expresses different
  genes distinguishing different cell types from
  one another and making them unique. DNA
  Microarrays allows us to look at many genes at
  once and determine which are expressed in
  different cell types.
• A microarray is a tool for analyzing gene
  expression that consists of a small membrane or
  glass slide containing samples of many genes
  arranged in a regular pattern

https//www.broadinstitute.org/files/news/stories/
full/OneChip.jpg
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How does it work?

1. Sample Cells are collected
2. RNA is extracted from samples
3. A solvent is used to dissolve sample separating
   DNA, Proteins, RNA, other cell parts.
4. Spin samples in vortex mixer to help dissolve
   sample
5. Place samples in centrifuge to separate the mRNA
   from other cell parts. RNA will be floating.
6. Remove the floating layer of RNA
7. Separate mRNA from by running sample through a
   column that sorts out mRNA.
8. mRNA attaches to Poly-T beads. Detach using a
   buffer that prevents hybridization

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• Make cDNA copy of mRNA and attach fluorescent
  label to cDNA
• A solution that contains the enzyme reverse
  transcriptase, poly-T primers, labeled
  nucleotides that have fluorescent molecule
  attached, is used to copy mRNA into DNA.
• A micro array is a slide covered with spots of
  single stranded DNA. Each spot represents a
  certain gene.

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• Place cDNA sample onto the microarray .
  Hybridization will occur where complementary DNA
  from different sources pair together to reform a
  double stranded DNA molecule.
• DNA that does not pair with DNA is washed off by
  submerging in a washing solution
• A Microarray scanner is used to produce an image
  and see where cDNA bound to the microarray.
• Results are shown in an image. The image will
  show information such as which genes are
  expressed in the cell type of original sample.

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How are Microarrays used?

• Microarray technology is used in cancer research
  to compare expressed genes in healthy cells and
  cancerous cells.
• Helps determine which genes are used in certain
  cell types and infer possible gene function
• To identify genes involved in the development of
  certain diseases

http//gunston.doit.gmu.edu/liverdisease/LindaImag
es/microarray2.jpg
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Pacific Biosciences

• SMRT DNA Sequencing Technology
• video
• Long Reads, Short Run Time, and High Quality Data
  at Lower Cost

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References

• Campbell, Neil A., and Jane B. Reece. Biology .
  Ed. Lisa A. Urry, et al. 8th      Edition ed.
  San Francisco Pearson Benjamin Cummings, 2008.
• Hartwell, Leland H., et al. Genetics From Genes
  to Genomes. 3rd Edition ed. New York
  McGraw-Hill, 2008. N. pag. Print.
• Canfield, Elizabeth. "Sanger Method for DNA
  sequencing ." Bio 111. Davidson University ,
  2001. Web. 30 Sept. 2009. lthttp//www.bio.davidson
  .edu/Courses/Bio111/seq.htmlgt.
• "Technology Demo." Pacific Biosciences Inc. .
  Pacific Biosciences, 2009. Web. 30 Sept. 2009.
  lthttp//www.pacificbiosciences.com/gt.