Enabling Technology: PCR and DNA microarray

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Enabling TechnologyPCR 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/PCRProcedur
e
Animation 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 gt 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 US10,000 from Cetus
• Cetus sold the patent to Hoffmann-La Roche for
  US300 M
• Patent 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 copies are available to insert into
the vectors. The recombinant DNA is then
replicated Inside 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 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
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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

• 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
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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 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
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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 effects
• Example 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 286531, 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
• YN
• 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