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Enabling Technology: PCR and DNA microarray

Enabling Technology: PCR and DNA microarray. CENG 109 Class 12. What is PCR (polymerase chain reaction)? . PCR is a method to make copies of a DNA sequence in a test tube. Essential to this reaction are: 1) an enzyme (DNA polymerase) 2) initiation templates (primers)

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Enabling Technology: PCR and DNA microarray

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  1. Enabling Technology:PCR and DNA microarray CENG 109 Class 12

  2. What is PCR (polymerase chain reaction)? • PCR is a method to make copies of a DNA sequence in a test tube. Essential to this reaction are: 1) an enzyme (DNA polymerase) 2) initiation templates (primers) 3) the control of temperature cycles. Picture credit: Royal Swedish Academy of Sciences

  3. How does PCR work? Picture: http://en.wikipedia.org/wiki/PCR#Procedure Animation: http://www.dnalc.org/home.html

  4. Why is PCR so powerful? • No. of theoretical maximum of double-stranded DNA after the nth cycle = 2n • PCR => logarithmic amplification • However, there is a limit of the length of DNA that can be amplified using this method (up to 10kb)

  5. Anecdotes on PCR invention • Kary Mullis was credited (not without controversy) for the PCR invention in 1983. He was awarded Nobel prize in 1993. • The concept of DNA replication outside living cells existed since 1971. But the real application cannot be realized until the use of Taq polymerase (a heat-stable enzyme) • Mullis received US$10,000 from Cetus Cetus sold the patent to Hoffmann-La Roche for US$300 M Patent lawsuit filed by Dupont was unsuccessful

  6. PCR applications • Forensic / identification tools (e.g. paternity test) • Diagnostic tools • Research tools • Enabling tools for recombinant DNA technology • Major supporting tool for genome sequencing

  7. Enabling rDNA applications PCR (step 2) is used to amplify a desired gene so that adequate copies are available to insert into the vectors. The recombinant DNA is then replicated Inside host cells (e.g. bacteria)

  8. Supporting genome sequencing • Accurate sequencing relies on good starting materials, a.k.a. pure DNA sequence in adequate quantities • PCR is used to • Provide fast amplification of short sequences that can be bracketed by primers • Aid in the amplification of DNA inside host cells

  9. Human Genome Project

  10. Human Genome Project Goals:  ■ identify all the approximate 30,000 genes in human DNA, ■ determine the sequences of the 3 billion chemical base pairs that make up human DNA, ■ store this information in databases, ■ improve tools for data analysis, ■ transfer related technologies to the private sector, and ■ address the ethical, legal, and social issues (ELSI) that may arise from the project. Milestones: ■ 1990: Project initiated as joint effort of U.S. Department of Energy and the National Institutes of Health ■ June 2000: Completion of a working draft of the entire human genome ■ February 2001: Analyses of the working draft are published ■ April 2003: HGP sequencing is completed and Project is declared finished two years ahead of schedule U.S. Department of Energy Genome Programs, Genomics and Its Impact on Science and Society, 2003

  11. What does the draft human genome sequence tell us? By the Numbers • The human genome contains 3 billion chemical nucleotide bases (A, C, T, and G).  • The average gene consists of 3000 bases, but sizes vary greatly, with the largest known human gene being dystrophin at 2.4 million bases.  • The total number of genes is estimated at around 30,000--much lower than previous estimates of 80,000 to 140,000.  • Almost all (99.9%) nucleotide bases are exactly the same in all people.  • The functions are unknown for over 50% of discovered genes. U.S. Department of Energy Genome Programs, Genomics and Its Impact on Science and Society, 2003

  12. What are we going to do with the information? • There are clear advantages (both in the social and business points of view) to speed up the use of this information.

  13. What is a DNA microarray? • A small solid support (e.g. glass, nylon or silicon) containing a large collection of single-stranded DNA in an array format • Also commonly known as gene chip, DNA chip, or biochip) • One of the key methods in making DNA chips originates from the same approach for making computer chips (photolithography)

  14. Fabrication • Fabrication via Printing • DNA sequence stuck to glass substrate • DNA solution pre-synthesized in the lab • Fabrication In Situ • Sequence “built” • Photolithographic techniques use light to release capping chemicals • 365 nm light allows 20-m resolution Slide Credit: Dr. Travis Doom, Department of Computer Science and Engineering Wright State University

  15. How does a DNA microarray work? • A DNA microarray is used to profile the expression of a large number of genes simultaneously. • The level of transcription of a certain gene is deduced by measuring the amount of hybridization of labeled RNA (or labeled cDNA) to a complementary probe. • DNA microarray animation

  16. Biotinylated RNA from experiment Each probe cell contains millions of copies of a specific oligonucleotide probe GeneChip expression analysis probe array Streptavidin- phycoerythrin conjugate Image of hybridized probe array

  17. DNA microarray applications • Gene hunters: detecting the presence and amount of a large number of specific genes • Provide diagnostics/prognostics (using probes containing disease-causing genes) • Choice Expressions: expression profiling of a large number of genes • Discover functions of genes • Get information about a large number of genes at the same time • Drug Discovery Tools: a low-risk, high-throughput approach • Use guilt-by-association approach to find the next hit • Red-flag candidates likely to cause side effects Example: Identify targets as anti-inflammatory drugs by comparing expressions of a collection of genes with IL-2 gene

  18. Example: on leukemia classification • Two forms of acute leukemia, ALL and AML, require different treatments for patients • DNA microarray experiment: examine expression of 6817 genes parallelly • Discover distinct genes associated with different types of acute leukemia Science 286:531, 1999

  19. How many genes can we study at one time ? • For a probe with length N consisting of Y subunits, the number of combinations • Y^N • For a singled stranded DNA probe with 25 bases, how many combinations are there? • How many steps are needed to do this with the Affymetrix technology? • Feature size: 100 micron in 1994  5 micron in 2005 • 10K array  100K array  500K array Information from www.affymetrix.com

  20. A status report of the market leader • Affymetrix is leading the DNA microarray market, based on its high-intensity platform • In 2003, the company has started to report net incomes. Over 1999-2003, total revenues trebled with a revenue of US$ 300M in 2003 • Partnership with big companies to develop new drugs (e.g. Millennium, GSK) and to integrate gene chip data into HIT (e.g. IBM) Information from DATAMONITOR

  21. Continued opportunites and… threats • New products for research and drug discovery • SNP chips • resequencing of disparate data from the genome project • Chips for basic research • Threats • Rising competition • Rapid innovations Information from DATAMONITOR

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