Saliterman, Fundamentals of BioMEMS and Medical Microdevices, Ch. 11
Genomics and
DNA Microarrays
Introduction to BioMEMS
MN-BIO4600 – Lecture 11
Genomics
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Understand the mechanism of DNA replication, protein synthesis,
gene expression, exchange and recombination of genetic material
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Understand endonucleases and DNA ligases capable of cutting
and rejoining DNA sequences at specific sites
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PCR and automatic DNA sequencing
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Bioinformatics (storing, analyzing, interpretation of data)
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Functional Genomics: assign biological function to genes, groups
of genes and particular gene interactions
Chromatin
Chromosomes
Fig 1:
Cycle of cell division
Chromosomes
Fig 1:
Human chromosomes
mother
father
Sex
chromosomes
Fig 1:
Human chromosomes
Genes
Fig 2:
The human chromosome
A piece of DNA that code for a
certain function in an organism
Structure of DNA
Fig 3:
Deoxyribonucleic acid (DNA)
3’
5’
Nucleotide Chain
Nucleotide
Hydrogen bonds
Fig 4:
Nucleotide chain
3’
5’
DNA Replication
DNA Replication
Fig 5:
DNA replication
Fig 5:
DNA replication
Polymerase Chain Reaction (PCR)
95C
60C
Fig 6:
PCR
72C
Fig 7:
PCR
72C
95C
60C
Fig 7:
PCR
https://www.youtube.com/watch?v=_YgXcJ4n-kQ
72C
Fig 8:
PCR
Distribution of DNAs with PCR
Fig 9:
PCR Products
https://www.youtube.com/watch?v=vK-HlMaitnE
DNA sequencing – Sanger Method
Used to identify the genetic
code of a DNA sample
(A) Based on the method of
PCR
(B) 4 different reaction
vessels
(C) radiolabelled nucleotides
(C) di‐deoxynucleotide
(adesine, guanine,
cytosine, thymine) that
terminates chain reaction
(D) Gel electrophoresis
DNA sequencing – Next Generation
Original DNA
Strands / code
Random
Fragmentation
«Shot gun»
DNA fragments
available for
PCR & sequencing
https://www.youtube.com/watch?v=LRnSNlilzJ8
DNA sequencing – Next Generation
Identification of the genetic code
of a DNA sample on chip
(A) DNA fragments immobilized
on beads
(B) Beads inserted into pH sensor
array (CMOS) 1.3 mill wells.
(C) Sequential wash of different
nucleotides
(D) Binding event associated with
the release of single protons
(H+)
Images courtesy of Ion Torrent
(E) Change in pH mapped as
binding of associated
nucleotide
Rothberg et al., Nature, 475, 2011, p. 348-352. doi:10.1038/nature10242
DNA sequencing – Next Generation
Mapping of
binding events to
one DNA sequence
Mapping of
Overlapping
DNA sequences
Calculation of chip size
Example:
We have a genome of 4 patients in a length of 10kBase
each. The required coverage is 500. Estimate the required
chip size if the chip loading is 80% and polyclonality is
40%?
How long time would it take to record the raw data
sequences given that the addition of each base pair takes
15 seconds?
Calculation of chip size
Throughput based on coverage
(# reads per reference bases = 500)
Compensation for polyclonality (40%)
Chip loading (80%)
Circular genome plot – 100kb intervals
Transcription
Transcription
Fig 10:
Transcription
Genetic Code
Stop
Start
Fig 11:
Messenger RNA Codon
https://www.youtube.com/watch?v=gG7uCskUOrA
Gene Expression (Protein Synthesis)
Fig 12:
Protein synthesis
Proteins
Fig 13:
Proteins
Expression
Gene Expression
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Active genes are being expressed through the production of mRNA.
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This production is monitored by microarrays that tell how much RNA
(if any) a gene is making.
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Based on the natural chemical attraction (hybridization) between
DNA on the array and RNA target molecules from the sample based
on complimentary base pairs.
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Only RNA target molecules that have exact complementary base pairs
will bind to the probes.
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Microarrays can measure the expression of every known human
gene.
Single Nucleotide Polymorphisms (SNP)
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DNA sequence variation occurring when a single nucleotide — A, T, C or G — in
the genome differs between members of a biological species or paired
chromosomes
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For example, a change in the sequence AAGCCTA to AAGCTTA, contain a
difference in a single nucleotide
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SNPs occur more frequently in non‐coding regions than in coding
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Exception: the SNP constitutes a more favorable genetic adaptation and is
implemented in the coding region (natural selection)
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May have major impact on health
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Scan the whole genome and look for genetic similarities among a group of
people who share the same disease
Pharmacogenomics
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How does the therapeutic and toxic response to drugs depend on the
individual genetic inheritance?
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Relationship between specific DNA sequence variation and drug
effect
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Administration of a given drug could result in different efficacy and
toxicity from one patient to the next
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Caused by the genetic variation in the form of single nucleotide
polymorphism that can alter RNA splicing and transcription between
individuals
Instrumentation
DNA Lab-on-a-Chip (LOC) Devices
 See also ch9: uTAS
100 mm
60 mm
DNA Microarray
Fig 14: Liu, RH, Analytical Chemistry, 76(7), 2004
DNA Microarrays
•
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Microarray analysis allows simultaneous study of genes (DNA sequences)
and gene products (mRNA and proteins)
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DNA may be obtained from any biological sample (same in all cells of the body)
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RNA must be obtained from specific tissue/fluid active in gene expression)
Two different formats:
cDNA microarrays
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Oligonucleotide microarrays types
•
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arrangement of “spots” of single stranded DNA probes (spot: 250m) printed onto a solid
matrix such as glass, nylon or silicon. Substrate size: < 4x4 cm
Fabricated probes of single stranded DNA in an exact sequence (spot: 11x11m) substrate
size 1.28x1.28 cm)
Applications:
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Gene expression
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Genotyping (single nucleotide polymorphisms ‐ SNP)
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Resequencing
Affymetrix GeneChip® Array
Fig 15:
Images courtesy of Affymetrix
Array Structure
Fig 16:
Gene chip (1,28x1,28 cm) and spots (11x11 m ) of DNA probes.
Fabrication of Microarrays
1) 5” square quartz wafer
naturally hydroxylated
- excellent substrate for attachment of linkers
used to attach probes on the assays
2) wafers placed in a bath of silane
- reacts with the hydroxylgroups of quartz
3) Linker molecules attached to silane
matrix provide surface that may be
spatially activated by light (mask)
4) availabe for nucleotide coupling
5) process repeated with old/new mask
- deprotection/coupling of new nucleotide
- terminated after 25 bp
6) Wafers deprotected and diced
- individual chips packaged in cartridge
- 49 to 400 different arrays
Fig 20:
Process flow of Affymetrix microarray chips (mask shown).
Probe Synthesis
Fig 22: Deprotection and coupling of nucleotides
https://www.youtube.com/watch?v=MuN54ecfHPw
Inspection, Dicing and Packaging
Fig 23:
Images courtesy of Affymetrix
Methodology
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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
•
Biotin
• water‐soluble B‐vitamin (vitamin B7)
• small size (MW 244 g/mol) means the biological activity of the nucleic
acid will most likely be unaffected upon binding
• Binds to streptavidin / avidin coated fluorescent probes
Hybridization
•
The sample is washed over the array for 14‐16 hours at 45C to allow
hybridization to occur.
– This process allows the chemical bonding of the DNA probes with the
matching RNA fragment.
Fig 17: Images courtesy of Affymetrix
Fluorescence Measurement
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Fluorescent dye washed over chip and binds to biotin
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Darker areas represent decreased expression, while lighter areas
represent increased expression:
Fig 18: Images courtesy of Affymetrix
Output
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The Affymetrix Scanner 3000 work station and GeneChip Array output
is data from an experiment showing the expression of thousands of
genes on a single GeneChip:
Fig 19: White spot indicate successful hybridiation.
Microarray Capability
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Measure tens of thousands of genes (activity) at the same time
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Obtain near‐comprehensive expression data for individual, tissues, or organs in
various states
•
Compare 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
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Detection of 1 RNA molecule (req. PCR) from 100,000 different RNAs
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Probes consists of 25 bases that represent a unique complimentary portion of
the target gene
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This short probe on the microarray measures expression of the complete gene
(1000’s bp long) by sampling only a small section of the gene
Summary
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Biomedical Micro Electro-Mechanical Systems
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Topics of study (curriculum):
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Introduction to BioMEMS
Principles of Biochemistry
Silicon and «Soft» Fabrication Techniques
Polymer Materials
Microfluidic Principles
Sensor Principles and Microsensors
Microactuators and Drug Delivery
Clinical Laboratory Medicine
Micro-Total-Analysis Systems
Detection and Measurement Methods
Genomics and DNA Microarrays
Proteomics and Protein Microarrays
Emerging BioMEMS technologies
Packaging, Power, Data, and RF Safety
Biocompatibility, FDA and ISO 10993
Thank you