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Enabling Technology:PCR and DNA microarray
CENG 109 Class 12
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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
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How does PCR work?
Picture: http://en.wikipedia.org/wiki/PCR#ProcedureAnimation: http://www.dnalc.org/home.html
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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)
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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 MPatent lawsuit filed by Dupont was unsuccessful
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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
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Enabling rDNA applications
PCR (step 2) is used to amplify a desired gene so that adequate copiesare available to insert into the vectors. The recombinant DNA is then replicatedInside host cells (e.g. bacteria)
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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
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Human Genome Project
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Human Genome ProjectGoals: ■ 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
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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
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• 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.
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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)
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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
FabricationFabrication
Slide Credit: Dr. Travis Doom, Department of Computer Science and EngineeringWright State University
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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
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Biotinylated RNAfrom experiment
GeneChip expressionanalysis probe array
Image of hybridized probe array
Each probe cell containsmillions of copies of a specific oligonucleotide probe
Streptavidin-phycoerythrinconjugate
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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 effectsExample: Identify targets as anti-inflammatory drugs by comparing expressions of a collection of genes with IL-2 gene
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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
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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
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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
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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