Introduction to BioMEMS & Medical Microdevices

DNA Microarrays and Lab-on-a-Chip Methods Companion lecture to the textbook: Fundamentals of BioMEMS and Medical Microdevices, by Prof. Steven S. Saliterman, http://saliterman.umn.edu/

Steven S. Saliterman

Deoxyribonucleic Acid “DNA”

Groton Public Schools

James D. Watson and Francis Crick, who discovered the structure of DNA in 1953.

Space-filling model of the DNA double helix.

Steven S. Saliterman

Chromosomes & Genes

Access Excellence @ the National Health Museum (left)

National Center for Biotechnology Information (right)

Chromosome 1

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Eukaryotic Gene Regulation

Evolution-textbook.org; Modified from Seet, BT et al. Nat. Rev. Mol. Cell. Biol. 7: 473-483, 2006

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Overview of Compounds Studied

Casado-Vela, J. Screening of protein-protein and protein-DNA inter-actions using microarrays: applications in biomedicine. Advances in Protein Chemistry and Structural Biology, Volume 95, 2014

Steven S. Saliterman

DNA Microarrays

Images courtesy of Affymetrix

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Array Structure

Images courtesy of Affymetrix

Steven S. Saliterman

Measurement of tens of thousands of genes at a time.

It is possible to obtain near-comprehensive expression data for individual, tissues, or organs in various states.

Comparisons are possible for transcriptional activity across different tissues in the same organism, across neighboring cells of different types in the same tissue, across groups of patients with and without a particular disease or with two different diseases.

Direct measurement of the activity of the genes involved in a particular mechanism or system.

Capability

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Formats

Microarray analysis allows simultaneous study of genes and gene products, including DNA, mRNA and proteins.

There are basically two formats: cDNA microarrays (clone-based):

A cDNA microarray is an orderly arrangement of “spots” of cDNA clones (from a library) printed onto a solid matrix such as glass, nylon or silicon.

Oligonucleotide-based microarrays : Photolithographic in situ synthesis. Used for gene expression, genotyping (single nucleotide

polymorphisms or SNPs), and resequencing.

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Resequencing

The Resequencing Array is used for identifying DNA in a sample. “Resequencing” means that when the DNA

is sequenced, it can be compared with an already established sequence database for the DNA of interest.

The probes that are used are made of segments of DNA 25 bases long, but the probes are used to basically sequence the DNA in the sample rather than identify a specific RNA or SNP.

Steven S. Saliterman

Gene expression microarrays are tools that tell how much RNA (if any) a gene is making.

They use the natural chemical attraction, or hybridization, between DNA on the array and RNA target molecules from the sample based on complimentary base pairs.

Only RNA target molecules that have exact complementary base pairs will bind to the probes.

Microarrays can measure the expression of every known human gene.

Gene Expression

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Single Nucleotide Polymorphisms

Small differences in a DNA sequence can have major impact on health.

Using microarrays, it is possible to scan the whole genome and look for genetic similarities among a group of people who share the same disease. A genotyping microarray may look for up to

100,000 SNPs or more. DNA samples from the patient may be obtained

from any biological sample, since DNA is the same in all cells of the body (unlike the RNA sampling for gene expression).

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RNA is extracted from a sample using PCR, allowing it to be more easily detected on the array. When the RNA is copied, biotin caps are

attached to each strand that will later act to bind fluorescent molecules that are washed over the array.

Note that different cells in the body have different amounts of RNA.

Oligonucleotide Microarray Methodology

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The sample is washed over the array for 14-16 hours to allow hybridization to occur. This process allows the chemical bonding of the

DNA probes with the matching RNA fragment.

Hybridization

Images courtesy of Affymetrix

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The amount of fluorescence in a given feature correlates with the amount of RNA that was present in the original sample.

If there is no fluorescence over a feature, then there is no matching RNA present. Lighter areas represent increased expression.

Image courtesy of Affymetrix

Fluorescence Measurement

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The Affymetrix Scanner work station and GeneChip Array output is data from an experiment showing the expression of thousands of genes on a single GeneChip:

Output

Images courtesy of Affymetrix

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Fabrication of Microarrays

Images courtesy of Affymetrix

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Silanation of Silicon Surface

Image courtesy of Affymetrix

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Probe Synthesis

Images courtesy of Affymetrix

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Inspection, Dicing and Packaging

Images courtesy of Affymetrix

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Polymerase Chain Reaction

An in vitro method of replicating small DNA sequences into millions of copies over a short period of time. Nanomolar quantities of DNA can be replicated within a

few hours. PCR may be used for genetic testing in disease

diagnosis, monitoring response to treatment, and tissue typing.

A typical PCR requires: Two oligonucleotide primers; A thermally stable DNA polymerase; Supply of free nucleotides; and A small amount of DNA sample that contains the

sequence of interest. The DNA fragment of interest must be known, so that short

DNA primer fragments can be synthesized in advance, and are complimentary to the 3´ end of each sample stand.

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Denaturing and Annealing Steps

A.

C.

B.

Carey, FA, Advanced Organic Chemistry 5th Ed., 2002

Desired sequence or target region to amplify.

Denaturing by heating 95 C

Annealing by cooling to 60 C, and binding of primers.

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D.

E.

Carey, FA, Advanced Organic Chemistry 5th Ed., 2002

Synthesis and Denaturing

The primer is extended in its 3” direction as it adds nucleotides that are complementary to the original DNA strand. End of Cycle 1

Denature again and prime.

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F.

G.

Carey, FA, Advanced Organic Chemistry 5th Ed., 2002

Elongation and Sequence Amplification

Elongation of the primed polynucleotide fragments completes the second cycle and gives 4 DNAs.

Among the eight DNAs formed in the third cycle are two having the structure shown. This structure increases disproportionately in the succeeding cycles.

End of Cycle 2

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DNA Amplification Results

Cycle Number

Total Number of DNAs

Number of DNAs Containing Only

the Target Region 0 1 0 1 2 0 2 4 0 3 8 2 4 16 8 5 32 22 10 1,024 1,004 20 1,048,566 1,048,526 30 1,073,741,824 1,073,741,746

Carey, FA, Advanced Organic Chemistry 5th Ed., 2002

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A CMOS IC qPCR LOC for POC

Norian, H. et al., An integrated CMOS qualitative-polymerase-chain-reaction lab-on-a-chip for point-of-care diagnostics Lab Chip, 2014, 14, 4076-4085

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Utilizes a Single-Photon Avalanche Diode…

Albert H. Titus, et al. CMOS Photodetectors in Photodiodes - World Activities in 2011. Ed. by Park, JW , pub. by InTech, 2011.

The cross-section of a SPAD CMOS sensor

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Heaters and Sensors

Norian, H. et al., An integrated CMOS qualitative-polymerase-chain-reaction lab-on-a-chip for point-of-care diagnostics Lab Chip, 2014, 14, 4076-4085

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Fully Encapsulated PCR Chip

Norian, H. et al., An integrated CMOS qualitative-polymerase-chain-reaction lab-on-a-chip for point-of-care diagnostics Lab Chip, 2014, 14, 4076-4085

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qPCR Thermal Cycling Profile

Norian, H. et al., An integrated CMOS qualitative-polymerase-chain-reaction lab-on-a-chip for point-of-care diagnostics Lab Chip, 2014, 14, 4076-4085

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Characterization of the SPAD

Norian, H. et al., An integrated CMOS qualitative-polymerase-chain-reaction lab-on-a-chip for point-of-care diagnostics Lab Chip, 2014, 14, 4076-4085

Steven S. Saliterman

Real-Time Quantitative PCR Data

Norian, H. et al., An integrated CMOS qualitative-polymerase-chain-reaction lab-on-a-chip for point-of-care diagnostics Lab Chip, 2014, 14, 4076-4085

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A PCR + Microarray Device

Petralia, S et al., In-Check system: a highly integrated silicon lab-on-chip for sample preparation, PCR amplification and microarray detection of nucleic acids directly from biological samples. Sensors and Actuators B 187 (2013) 99-105.

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Lab-on-a-Chip Cross-Section

Petralia, S et al., In-Check system: a highly integrated silicon lab-on-chip for sample preparation, PCR amplification and microarray detection of nucleic acids directly from biological samples. Sensors and Actuators B 187 (2013) 99-105.

Steven S. Saliterman

Process

Petralia, S et al., In-Check system: a highly integrated silicon lab-on-chip for sample preparation, PCR amplification and microarray detection of nucleic acids directly from biological samples. Sensors and Actuators B 187 (2013) 99-105.

Steven S. Saliterman

Summary

(1) Chromosomes, genes and gene regulation.

(2) DNA Microarrays (3) Methodology (4) Fabrication (5) Polymerase Chain Reaction (6) A CMOS IC qPCR LOC for POC (7) A PCR + Microarray Device