Saliterman, Fundamentals of BioMEMS and Medical Microdevices, Ch.

11

Genomics and
DNA Microarrays
Introduction to BioMEMS
MN-BIO4600 Lecture 11
2015
 Genomics
The biochemical
processes for making DNA
exchange of genetic information
the process by that
enzymes which information breaking andjoining
rejoining thestrands
of DNA
of process of copying
strands together byacatalyzing
between twocleave
DNA the phosphodiester
molecules
from abond
genewithin
is used in biochemistry
a polynucleotide to forms newthe
chain molecules double-stranded
formationofof
DNA DNA molecule
a phosphodiester bond.

Understand the mechanism of DNA replication, protein synthesis,

gene expression, exchange and recombination of genetic material

Understand endonucleases and DNA ligases capable of cutting

and rejoining DNA sequences at specific sites
process of determining the
PCR and automatic DNA sequencing precise order of nucleotides
within a DNA molecule

Bioinformatics (storing, analyzing, interpretation of data)

Functional Genomics: assign biological function to genes, groups

of genes and particular gene interactions
The polymerase chain reaction - generates thousands to millions of
copies of a particular DNA sequence from one or two single strands.
 Chromatin

Chromosomes
Fig 1: Cycle of cell division
 Chromosomes

Fig 1: Human chromosomes

mother
father

Sex
chromosomes

Fig 1: Human chromosomes

 A piece of DNA that code for a
Genes certain function in an organism

Fig 2: The human chromosome

 Structure of DNA

Fig 3: Deoxyribonucleic acid (DNA)

 3
5
Nucleotide Chain

Nucleotide

Hydrogen bonds 5

Fig 4: Nucleotide chain

3
DNAReplication
DNA Replication

Fig 5: DNA replication

Fig 5: DNA replication
Polymerase Chain Reaction (PCR)

95C

60C

Fig 6: PCR
 72C

Fig 7: PCR
 72C

95C

60C

Fig 7: PCR
 72C

Fig 8: PCR
Distribution of DNAs with PCR

Fig 9: PCR Products

DNA sequencing

Usedtoidentifythegenetic
codeofaDNAsample
(A) Basedonthemethodof
PCR
(B)4differentreaction
vessels
(C)radiolabelled nucleotides
(C)dideoxynucleotide
(adesine,guanine,
cytosine,thymine)that
terminateschainreaction
(D)Gelelectrophoresis
Transcription
Transcription

Fig 10: Transcription

 Genetic Code
Stop

Start Fig 11: Messenger RNA Codon

Gene Expression (Protein Synthesis)

Fig 12: Protein synthesis

 Proteins

Fig 13: Proteins

Instrumentation
DNA Lab-on-a-Chip (LOC) See also ch9:
uTAS
Devices 60 mm

100 mm
DNA Microarray
Fig 14: Liu, RH, Analytical Chemistry, 76(7), 2004
 DNA Microarrays
Microarray analysis allows simultaneous study of genes (DNA
sequences) and gene products (mRNA and proteins)
DNAmaybeobtainedfromanybiologicalsample(sameinallcellsofthebody)
RNAmustbeobtainedfromspecifictissue/fluidactiveingeneexpression)

Two different formats:

cDNA microarrays
arrangementofspots ofsingle stranded DNAprobes(spot:250m)printedonto
asolidmatrixsuchasglass,nylonorsilicon.Substratesize:<4x4cm
Oligonucleotide microarrays types
Fabricatedprobesofsinglestranded DNAinanexactsequence(spot:11x11m)
substratesize1.28x1.28cm)

Applications:
Geneexpression
Genotyping(singlenucleotidepolymorphisms SNP)
Resequencing
 Single Nucleotide Polymorphisms (SNP)

DNAsequencevariationoccurringwhenasinglenucleotide A,T,
CorG inthegenomediffersbetweenmembersofabiological
speciesorpairedchromosomes
Forexample,achangeinthesequenceAAGCCTAtoAAGCTTA,
containadifferenceinasinglenucleotide
SNPs occurmorefrequentlyinnoncodingregionsthanincoding
Exception:theSNPconstitutesamorefavorablegeneticadaptation
andisimplementedinthecodingregion(naturalselection)
Mayhavemajorimpactonhealth
Microarrays:scanthewholegenomeandlookforgenetic
similaritiesamongagroupofpeoplewhosharethesamedisease
Agenotypingmicroarray maylookfor100,000SNPs ormore.
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.
Expression
 Gene Expression

Geneexpressionmicroarrays aretoolsthattellhowmuchRNA(if
any)ageneismaking.
Theyusethenaturalchemicalattraction,orhybridization,between
DNAonthearrayandRNAtargetmoleculesfromthesamplebased
oncomplimentarybasepairs.
OnlyRNAtargetmoleculesthathaveexactcomplementarybasepairs
willbindtotheprobes.
Microarrays canmeasuretheexpressionofeveryknownhuman
gene.
 Microarray Capability
Measuretensofthousandsofgenes(activity)atthesametime
Obtainnearcomprehensive expressiondataforindividual,tissues,or
organsinvariousstates
Comparetranscriptionalactivityacrossdifferenttissuesinthesame
organism,acrossneighboringcellsofdifferenttypesinthesame
tissue,acrossgroupsofpatientswithandwithoutaparticulardisease
orwithtwodifferentdiseases
Detectionof1RNAmolecule(req.PCR)from100,000differentRNAs
Probesconsistsof25bases thatrepresentauniquecomplimentary
portionofthetargetgene
Thisshortprobeonthemicroarray measuresexpressionofthe
completegene(1000sbp long)bysamplingonlyasmallsectionofthe
gene
 Methodology

RNAisextractedfromasampleusingPCR,allowingittobemore
easilydetectedonthearray
WhentheRNAiscopied,biotin capsareattachedtoeachstrandthatwill
lateracttobindfluorescentmoleculesthatarewashedoverthe array
NotethatdifferentcellsinthebodyhavedifferentamountsofRNA

Biotin
watersolubleBvitamin(vitaminB7)
smallsize(MW244g/mol)meansthebiologicalactivityofthenucleic
acidwillmostlikelybeunaffecteduponbinding
Bindstostreptavidin /avidin coatedfluorescentprobes
 Hybridization
Thesampleiswashedoverthearrayfor1416hoursat45Ctoallow
hybridizationtooccur.
ThisprocessallowsthechemicalbondingoftheDNAprobeswiththe
matchingRNAfragment.

Fig 17: Images courtesy of Affymetrix

 Fluorescence Measurement
Fluorescentdyewashedoverchipandbindstobiotin
Darkerareasrepresentdecreased expression,whilelighterareas
representincreased expression:

Fig 18: Images courtesy of Affymetrix

 Output
TheAffymetrix Scanner3000workstationandGeneChip Array
outputisdatafromanexperimentshowingtheexpressionof
thousandsofgenesonasingleGeneChip:

Fig 19: White spot indicate successful hybridiation.

 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

Inspection, Dicing and Packaging

Fig 23: Images courtesy of Affymetrix

 Pharmacogenomics

Howdoesthetherapeuticandtoxicresponsetodrugsdependon the
individualgeneticinheritance?
RelationshipbetweenspecificDNAsequencevariationanddrug
effect
Administration ofagivendrugcouldresultindifferentefficacyand
toxicityfromonepatienttothenext
Causedbythe geneticvariationintheformofsinglenucleotide
polymorphismthatcanalterRNAsplicingandtranscriptionbetween
individuals
 Summary

Biomedical Micro Electro-Mechanical Systems

Topics of study (curriculum):
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