MICROCHIP TECHNOLOGY FOR DISEASEIDENTIFICATIONMicroarray consists of thousands of microscopicspots that are attached to solid spot.In microarray these spots are calledprobes .Probes can be single stranded DNA in case of DNA microarray thathybridize with target labelled with dye. Upon hybridization,a signal is emittedby labelled fluorescent dye.Classification of microarray founded on the mode ofpreparationOn the basis of the mode of preparation of thearray, microarrays are classified into three types:· The spotted arrayon glass: Spotted arrays are those arraysthat are prepared on poly-lysine coated glass microscope slides.
This allowsbinding of high-density DNA by means of slotted pins. It also permitsfluorescent labeling of the sample.· Self-assembledarrays: Self assembled arrays are fiberoptic arrays that are synthesized by the deposition of DNA prepared on smallpolystyrene beads. The beads are fixed on the etched ends of the fiber opticarray. Different DNA can be prepared on different beads.
· In-situ synthesized arrays: These arraysare prepared by chemical synthesis on a solid spot. These arrays are used for expressionanalysis, sequencing and genotypingClassification ofmicroarray on basis of probeThese are given belowDNA microarrays: Gene chip,Biochip and DNA chip are other names for DNA microarrays. Single stranded DNAprobe used in DNA chip. DNA microarrays are classified on four types that arev DNA microarraysv oligo DNA microarraysv BAC microarrays v SNP microarrays · MMChips: MMchip are used for studying the interactions between DNA andprotein. Two commonly techniques used are ChIP-chip (Chromatinimmunoprecipitation (ChIP) followed by array hybridization) and ChIP-seq (ChIPfollowed by massively parallel sequencing) .· Proteinmicroarrays: it allows classification ofhundreds of thousands of proteins in a highly parallel way.
.· Peptidemicroarrays: These are used to study protein–protein interactions.Tissue microarrays· Cellularmicroarrays: these microarrays allows screening of genomic libraries and also used toinvestigate the local cellular microenvironment.· Chemical compoundmicroarrays: this is used for drug screeningand drug discovery. This microarray is most common technique that used inpharmaceutical industry · Antibodymicroarrays: they contain antibodies onsolid spot and allows detection of antigens Basic principle of DNA CHIP: · DNA microarraymaking· Hybridization· Results delivery Microchip Technologyfor disease identification:DNA chip allows researcher not only to study unique single aspect of the host/pathogen relationshipbut also allow scientists to discoverand understand disease pathways thus develop improved means of detection,treatment.Furthermore, DNA chip also helps to monitor whole-genome host andpathogen gene expression thus providedetailed study of the progression of an infectious disease.DNA microchip detectinfectious diseases by determine the genes involved in disease and pathogenicmicrobes . DNA microchip gives rapid, reproducible,precise, efficient ,reliable and high-throughput screening of disease anddisease causative agents.
That is why microarray is preferred for diagnosis ofdiseases.For treatment of patients rapid diagnosis of disease is veryimportant.Performance of diagnosis by DNAmicroarray is improving by integration of other tools.
DNA microarray is goldstandard method for diagnosis of infectious diseases caused by pathogenicmicrobes. Microarray is one of the latest technique that isbeing used for cancer research.Microchip provides help in pharmacologicalmethodology to treat several diseases which include oral lesions. Microchiphelps in identifying large amount of samples which can be old or new samples.
It can help to exam the occurrence of a specific marker in tumors.Recently use of microarray in dentistryhas been very limited but in future when DNA microchip technology will beaffordable, there will be increase in its use. Here, we discuss the varioustechniques and applications of microarray or DNA chipPathogens that cause infectiousdiseases can be detected by using microarray.Infectious diseases cause seriousdeath such as virus causes AIDS,avian influenza,dengue fever. Rapid detectionof pathogenic microbes is important to treat the patients.DNA microarray detectpathogenic microbes not only by increasing the assay capabilities i.esensitivity and specificity but also allow high throughput detection ofmicrobial pathogens.
.Disease diagnosed by microarray arecancer,chronic inflammatory diseases,crohn’s disease,type 1 diabetes,ulcerativecolitis,alzheimers disease.Summary of diseases that diagnosed by microarray isgiven below DNA MICROCHIP FOR THE DIAGNOSIS OF INFECTIOUSDISEASES· Infectitiousdiseases by different speciesInfectitious diseases are very seriousand human beings are continuously fighting to avoid pathogens. Due to newpathogen emergence many infectitious diseases are spreading by viruses like immune deficiency syndrome(AIDS), by bacteria like avianinfluenza, malaria, measles and also by antibioticresistance superbacteria like methicillin-resistant Staphylococcusaureus vancomycin resistanct S.
aureus.· Reasonsof difficulty in identification of pathogensDue to abuse of antibiotics, overuse ofthe immunosuppressants in transplantation and overdose of drugs as anticancertherapy it is getting impossible for the for the rapid detection andidentification of the pathogenic agents.· Differentmethods for identification of pathogenAlthough various methods have made likenucleic acid assay (e.g. PCR based assay), culture based assay ( in whichantibodies are being administrated and serological and immunological methodsbut these methods has some limitations like low speed intensive labor andresulting in high fast false results.· Reasonwhy Microchip technology prevailedWhereas, now Microchip technology hasbeen developed and successfully detected the microbial pathogens. it providesidentification of pathogens with increased assay capabilities like highsensititivity specificity , high throughput detection thus disease is nowpossible to be identified with greater ease and fastly so that better decisionscan be made.Pathogen detectionand identificationMost important application ofmicrochip is identification of infected pathogen.
As pathogens show diversegenetic composition, microarray is ideal tool to examine all gene sequences. Multi-pathogen identification microchip ( MPID) is used to identify pathogenic eukaryotes,viruses andparokaryotes. Researchers apply the specific regions of DNA frommicro-organisms using PCR and then used multi- pathogenidentificationmicroarray to identify the absence and presence of pathogen specific DNAsequences.It detects 10 femptograms of pathogenic DNA.Microarray alsodistinguish different strains of mycobacterium tuberculosis andP.aeruginosa.Microarray detect 16S ribosomal DNA sequence for identification ofmycobacterium tuberculosis.
PATHOGEN DETECTION ARRAY DESIGNMany groups have been applied microarray technology to pathogen idenification.Their approaches can be different according to the range of pathogens that are target, the probe design approachand array platform used. ViroChip:It was the first microarray designed foridentification of many pathogens.ViroChip has 1600 probes that derived from the140 complete viral genomes accessible in GenBank when array was designed. Laterversions of the array were established that cover a wide range of viruses asfurther genomes were published.
The ViroChip is made-up by mechanicallyspotting produced oligonucleotides on glass slide. The oligos are 70-mers thatare selected to match sequences that are common to a taxonomic family but notpresent in other families. For some families, oligos were designated at thegenus level. As the probes are designed against conserved sequences, theViroChip can be used to identify new viruses within the same family as afamiliar, sequenced virus.Advantages of virochipViroChip platform are the low cost of spotted oligo arrays and it candetect novel viruses within a known family.
Disadvantages ofvirochipIhe disadvantages of virochip can result mostly from the limitations ofspotted oligo technology.The low density of probes can be spotted on eachslide.It is an expensivetechnology. The latest version of the ViroChip solves these issues followingthe use of the Agilent ink-jet array . Probe designs have to be updatedoccasionally to remain in progress with novel genomes in GenBank.
GreeneChip for pathogen detectionThe ‘GreeneChip’arrays show a broader-spectrum approach to pathogen identification .These chipsare high-density oligonucleotide arrays that are fabricated by using theAgilent inkjet system. GreeneChipVr version 1.0 has 9477 probes for virusinfecting vertebrate. GreeneChipPm v1.0 is a panmicrobial array design thatcontain all of GreeneChipVr probes alongwith probes for several thousand pathogenic bacteria, protozoa and fungi thuscontaining total of 29?495 60-mer oligos. Due to the use of long (60-mer)oligos sensitivity of this microarray is compareable to virochip.It issensitive technique for virus identification.
DNA Microchip For The Diagnosis Of INFECTIOUSDISEASES· Infectitiousdiseases by different speciesInfectitious diseases are very seriousand human beings are continuously fighting to avoid pathogens. Due to newpathogen emergence many infectitious diseases are spreading by viruses like immune deficiency syndrome(AIDS), by bacteria like avianinfluenza, malaria, measles and also by antibioticresistance superbacteria like methicillin-resistant Staphylococcusaureus vancomycin resistanct S. aureus.· Reasonsof difficulty in identification of pathogensDue to abuse of antibiotics, overuse ofthe immunosuppressants in transplantation and overdose of drugs as anticancertherapy it is getting impossible for the rapid detection and identification ofthe pathogenic agents.· Differentmethods for identification of pathogenAlthough various methods have made likenucleic acid assay (e.
g. PCR based assay), culture based assay ( in whichantibodies are being administrated and serological and immunological methodsbut these methods has some limitations like low speed intensive labor andresulting in high fast false results.· Reasonwhy Microchip technology prevailedWhereas, now Microchip technology hasbeen developed and successfully detected the microbial pathogens. It providesidentification of pathogens with increased assay capabilities like highsensititivity specificity, high throughput detection thus disease is nowpossible to be identified with greater ease and fastly so that better decisionscan be made.Detection of Pathogenic Viruses by MicrochiptechnologyA virus is small infectious agentthatinvades living cell to replicate. Viral infection causes many symptoms likesore throat, pain in body, common cold.
Viral infection can be very dangerousto the community because disease caused by viruses undergo virals and fastcontagiousness.Identification of different virus using differentassayIt is necessary to get protection againviral diseases faster.Viruses contain specific nucleic acid sequences that are 30% different in the same species oreven several types of serotypes exits.
So ,it is not very difficult to detectpathogens by nucleic acid-assays.Hepatitisvirus identification by Quantitative nano gold-silver stain Microchip Hepatitis virus cause chronic liverdisease and hepatocellular carcinoma. It is being detected from a patient’sserum by quantitative nano gold-silver stain microchip. The use of gold nanoparticles treated with silver (AgNO3) for signalamplification has increased sensitivity, thus allowing detection of viruse upto100fM of ampicon. Detection of the Human papilloma virusHuman papilloma virus (HPV) has almost120 different types which infectsthrough the skin and mucous membranes, also causes cervical cancer.HPV detection chipResearchers reported several specifictypes of probes for HPV detection.
Yoon and his coworkers developed microchipfor the detection of the HPV known as HPV detection chip then it was furthermodified by Moon and his coworkers this Microchip having dual probes for the identification of 40 types of HPV. This assayprovides 100% sensitivity and 98% of the specificity relatively much betterresults are obtained.Probes were designed on the basis of thesequence of the L1, E6 and E7 genes of almost 35 types of HPV virus thus arecomplementarity to the sequence of probe. Result 35 types of genotypes of HPV wereobtained from cervical cell species of 4898 Korean adult women and 68 cervicalcancer cases. Thus different disease can be diagnose and identified with greater ease than theconventional methods..Identification of the Genetic diseases by MicrochipTechnologyGenetic disorders are known to be ashereditary disease due to inherited gene that causes abnormalities or mutationsand ultimately alters the functions of the normal gene and passes to nextgenerations resulting that particular disease in new born.
Classification ofgenetic disorders Genetic disorders are classified into I. Chromosomal abnormalities II. Mutationsin DNA sequence 1. Chromosomal Abnormalities Detection byMicrochip “Genetic disorders that causechromosomal abnormality by Duplication,Deletion, Translocation or Inversion of chromosomal part and results inabnormality of chromosomes.
“Several different diseases are causeddue to chromosomal abnormalities like · Down’s syndrome ( withchromosome 21)· Edwards syndrome (withchromosome 18)· Patau syndrome (withchromosome 13) · Turner syndrome (withchromosome XO)· Klinefelter syndromewith having chromosome (XXY)Microchip for the identification of diseases caused by chromosomal abnormalitiesand diagnosisFor all the chromosomal abnormalitieswhich causes diseases can be identified by the fabrication of the microchipcomprising specific chromosome relatedBAC chip which plays important role in diagnose of abnormalities detectedand it can be reconfirmed by a method known as fluorescencein situ hybridization (FISH). Fluorescencein situ hybridization (FISH)”FISH is amethod by which the position of specific DNA sequence present on chromosomescan be located. Most of the situhybridization methods use fluorescentprobes for the identification of specific DNA sequence.” Ø Successful in situ hybridization method based onthe stability of the DNA doublehelix. Principles of fluorescence in situ hybridization (FISH) (a) Important elementsneed to perform fish method is DNA probe and a target sequence.
(b) DNA probe I labelled by methods randomprimer labeling nick translation, and different types of PCR reactions before hybridization. (c) Labelling is done by two strategies: Indirect labelling method (left side) Direct labelling method (right side) Indirect labeling Indirect labeling, probes are labeled by modified nucleotides that containing hapten Direct labeling Direct labeling uses nucleotides that have been directly modified to contain a Fluorophore. (d) Then the labeled probe and the target DNA aredenatured at specific temperatures. (e) By the combination of the denatured probe and specifictarget sequence annealing of the complementary DNA sequence is done.Ø In indirect labeling of the probe anextra step is to be done for the visualization of the hepten that are not fluorescent and thus can be use in thedifferent immunological detection systems. Advantagesof Different labeling Probe in Identification Directly labeled probes Indirect labeled probe Ø By direct labeling FISH method can be done faster.
Ø Probes provide signal amplification by using various layers of antibodies. Ø Signals that are given are more brighter compared to background levels. Mutations in the DNA sequence “Mutationin the sequence of the DNA is due to change in the amino acid sequence ,splicing at different sites and ultimately lack of the protein produced thatcause different disease phenotypes.”Identification of mutation and diagnosis bymicrochip Geneticcounselling is themethod in which entire genome of the patients or relatives having risk ofinherited disorders is sequenced to know about the nature, probability,developing and transmitting of disease so that important decisions like type oftreatment or in family planning can be made Ø Microchipsare used for diagnosis and identification of many diseases using classicalmethod known as genetic counselling, SNPs detectionMicrochip plays an important role inidentification of diseased genes, particular those genes causing differentmutations in stages of life.Example Ø Cardiovasculardisease id diagnose by the identification of multiple single nucleotide known as polymorphismsin disease-relevant genes, results in the different patterns of genotypes. Detection of SNPs in Four Steps By Microchip1.
Hybridization2. Cleavageof present mismatch sites3. Todenture the hybridized Dimer4. Detectionof four different probes with dsDNAIdentification of Mutations Caused by Monogenic DisordersMonogenicDisorders are also known as Single Abnormal Gene and there is a singledefective gene present on autosomes.· Autosomaldominant diseasesAvellinoCorneal dystrophy is due to mutation occurring in gene that is coding for?IG-H3 which is autosomal dominant diseasescan be homozygous mutation and heterozygous mutation. It starts with blurrysymptom in centre of the cornea and ends up with vision loss with aging.Method of identification of disease by usingMicrochip Microchip for the cure of the cornealdystrophy can be used for detecting the mutations in Exons 4 and 12 of the?IG-H3. Conditionsprovided to Microchip Ø Microchipwas optimized at very high specificity and sensitivityØ Capturedprobe concentration should be at range of (100, 50, 30, and 10 µM),Ø Thelength of probe should be of 11 to 17polymersØ Hybridizationtime given should be (1, 2, 3, 4, 6, and 24 hrs.
). Method ResultCheuguinvented a complementary DNA microchipthat allows identification of different genetic mutation present in theheterozygous mutation carriers and normal patient.Above performed chip method used bloodsamples of 98 patients that give excellent result with 100% of accuracy.Cancer identificationby microchip Microchips initially used by Martin andhis colleagues for the identification of the 170 gene expressed differently inthe present of breast tumour cells. Thendifferent rarer cancer cells like · Skin cancer cells,· salivary gland cancer,· sarcoma cancerous cells ,· small cell lung cancer· small intestine cancer cells wereidentified by the use of microchip Method of identification of rare cancer cells Future prospectiveregarding to the Microchip in disease identification · ModificationsFor its increase in the practical use ofthe Microchip it is being making affordable, convenient to practice, accurateand portable.
. ExampleSuchlateral flow assay should provide diagnosis of many diseases in clinical andenvironment sample by on site pathogen and SNP detection· Detectionof chromatin By combining of microchip assay withdifferent kinds of other techniques it is making possible to detect evenchromatin level changes.· GenomeprojectsMany genome projects and infectiousorganisms are providing large amount of information about genome sequencing andthus by using probes many different diseases at gene level are making possibleto identify and diagnose easily.· Smartdrugs With advancement in microchip technologymultiple genetic markers and their functions will be identified in short futureand then this information will be used by medical researchers to produce anddiagnose patients with smart drugs.