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#ProcedureAnimation: http://www.dnalc.org/home.html

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)

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

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 copiesare available to insert into the vectors. The recombinant DNA is then replicatedInside 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 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

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 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)

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

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 RNAfrom experiment

GeneChip expressionanalysis probe array

Image of hybridized probe array

Each probe cell containsmillions of copies of a specific oligonucleotide probe

Streptavidin-phycoerythrinconjugate

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

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