Download 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)
3) the control of
temperature cycles.
Picture credit: Royal Swedish Academy of Sciences
How does PCR work?
Picture: http://en.wikipedia.org/wiki/PCR#Procedure
Animation: http://www.dnalc.org/home.html
Why is PCR so powerful?
No. of theoretical
maximum of doublestranded 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)
•
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 heatstable 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
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
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)
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
Human Genome Project
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
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
• 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.
What is a DNA microarray?
• A small solid support (e.g. glass, nylon or
silicon) containing a large collection of singlestranded 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)
Fabrication


Fabrication via Printing
 DNA sequence stuck
to glass substrate
 DNA solution presynthesized 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
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
Biotinylated RNA
from experiment
GeneChip expression
analysis probe array
Image of hybridized probe array
Each probe cell contains
millions of copies of a specific
oligonucleotide probe
Streptavidinphycoerythrin
conjugate
DNA microarray applications
•
Gene hunters: detecting the presence and amount of a large
number of specific genes
– Provide diagnostics/prognostics (using probes containing diseasecausing 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
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
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
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
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