MICROCHIP slotted pins. It also permits fluorescent labeling

MICROCHIP TECHNOLOGY FOR DISEASE
IDENTIFICATION

Microarray consists of thousands of microscopic
spots that are attached to solid spot.In microarray these spots are called
probes .Probes can be single stranded DNA in case of DNA microarray that
hybridize with target labelled with dye. Upon hybridization,a signal is emitted
by labelled fluorescent dye.

Classification of microarray founded on the mode of
preparation

On the basis of the mode of preparation of the
array, microarrays are classified into three types:

·        
The spotted array
on glass: Spotted arrays are those arrays
that are prepared on poly-lysine coated glass microscope slides. This allows
binding of high-density DNA by means of slotted pins. It also permits
fluorescent labeling of the sample.

·        
Self-assembled
arrays: Self assembled arrays are fiber
optic arrays that are  synthesized  by the deposition of DNA prepared on small
polystyrene beads. The beads are fixed on the etched ends of the fiber optic
array. Different DNA can be prepared on different beads.

·        
In-situ synthesized arrays: These arrays
are prepared by chemical synthesis on a solid spot.  These arrays are used for expression
analysis, sequencing and genotyping

Classification of
microarray on basis of probe

These are given below

DNA microarrays: 

 Gene chip,
Biochip and DNA chip are other names for DNA microarrays. Single stranded DNA
probe used in DNA chip. DNA microarrays are classified on four types that are

v  DNA microarrays

v   oligo DNA microarrays

v  BAC microarrays 

v  SNP microarrays

 

·        
MMChips: MMchip are used for studying the interactions between DNA and
protein. Two commonly techniques used 
are  ChIP-chip (Chromatin
immunoprecipitation (ChIP) followed by array hybridization) and ChIP-seq (ChIP
followed by massively parallel sequencing) .

·        
Protein
microarrays: it allows classification of
hundreds of thousands of proteins in a highly parallel way..

·        
Peptide
microarrays: These are used to study  protein–protein interactions.

Tissue microarrays

·        
Cellular
microarrays: these microarrays allows screening  of genomic libraries and also used to
investigate the local cellular microenvironment.

·        
Chemical compound
microarrays: this is used for drug screening
and drug discovery. This microarray is most common technique that used in
pharmaceutical industry

·        
Antibody
microarrays: they contain antibodies on
solid spot and allows detection of antigens

Basic principle of DNA CHIP:

·        
DNA microarray
making

·        
Hybridization

·        
Results delivery

 

 

 

 

 

 

Microchip Technology
for disease identification:

DNA chip allows  researcher 
not only to study unique single aspect of the host/pathogen relationship
but also allow  scientists to discover
and understand disease pathways thus develop improved means of detection,treatment.

Furthermore, DNA chip also helps to monitor whole-genome host and
pathogen gene expression thus  provide
detailed study of the progression of an infectious disease.DNA microchip detect
infectious diseases by determine the genes involved in disease and pathogenic
microbes .

 

 

 

 

DNA microchip gives rapid, reproducible,
precise, efficient ,reliable and high-throughput screening of disease and
disease causative agents.That is why microarray is preferred for diagnosis of
diseases.For treatment of patients rapid diagnosis of disease is very
important.

Performance of diagnosis by DNA
microarray is improving by integration of other tools.DNA microarray is gold
standard method for diagnosis of infectious diseases caused by pathogenic
microbes. Microarray is one of the latest technique that is
being used for cancer research.Microchip provides help in pharmacological
methodology to treat several diseases which include oral lesions. Microchip
helps 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 dentistry
has been very limited but in future when DNA microchip technology will be
affordable, there will be increase in its use. Here, we discuss the various
techniques and applications of microarray or DNA chip

Pathogens that cause infectious
diseases can be detected by using microarray.Infectious diseases cause serious
death such as virus causes AIDS,avian influenza,dengue fever. Rapid detection
of pathogenic microbes is important to treat the patients.DNA microarray detect
pathogenic microbes not only by increasing the assay capabilities i.e
sensitivity and specificity but also allow high throughput detection of
microbial pathogens..

Disease diagnosed by microarray are
cancer,chronic inflammatory diseases,crohn’s disease,type 1 diabetes,ulcerative
colitis,alzheimers disease.Summary of diseases that diagnosed by microarray is
given below

            

 

 

DNA MICROCHIP FOR THE DIAGNOSIS OF INFECTIOUS
DISEASES

·       
Infectitious
diseases by different species

Infectitious diseases are very serious
and human beings are continuously fighting to avoid pathogens. Due to new
pathogen emergence many infectitious diseases are spreading by viruses like immune deficiency syndrome
(AIDS), by bacteria like avian
influenza, malaria, measles and also by antibiotic
resistance superbacteria like methicillin-resistant Staphylococcus
aureus  vancomycin resistanct S. aureus.

·       
Reasons
of difficulty in identification of pathogens

Due to abuse of antibiotics, overuse of
the immunosuppressants in transplantation and overdose of drugs as anticancer
therapy it is getting impossible for the for the rapid detection and
identification of the pathogenic agents.

·       
Different
methods for identification of pathogen

Although various methods have made like
nucleic acid assay (e.g. PCR based assay), culture based assay ( in which
antibodies are being administrated and serological and immunological methods
but these methods has some limitations like low speed intensive labor and
resulting in high fast false results.

·       
Reason
why Microchip technology prevailed

Whereas, now Microchip technology has
been developed and successfully detected the microbial pathogens. it provides
identification of pathogens with increased assay capabilities like high
sensititivity specificity , high throughput detection thus disease is now
possible to be identified with greater ease and fastly so that better decisions
can be made.

Pathogen detection
and identification

Most important application of
microchip is identification of infected pathogen. As pathogens show diverse
genetic composition, microarray is ideal tool to examine all gene sequences.

Multi-pathogen identification microchip ( MPID) is used to identify pathogenic eukaryotes,viruses and
parokaryotes. Researchers apply the specific regions of DNA from
micro-organisms using PCR and then used multi- pathogenidentification
microarray to identify the absence and presence of pathogen specific DNA
sequences.It detects 10 femptograms of pathogenic DNA.Microarray also
distinguish different strains of mycobacterium tuberculosis and
P.aeruginosa.Microarray detect 16S ribosomal DNA sequence for identification of
mycobacterium tuberculosis.

PATHOGEN DETECTION ARRAY  DESIGN

Many 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 approach
and  array platform used.

ViroChip:

It was the first microarray designed for
identification of many pathogens.ViroChip has 1600 probes that derived from the
140 complete viral genomes accessible in GenBank when array was designed. Later
versions of the array were established that cover a wide range of viruses as
further genomes were published. The ViroChip is made-up by mechanically
spotting produced oligonucleotides on glass slide. The oligos are 70-mers that
are selected to match sequences that are common to a taxonomic family but not
present in other families. For some families, oligos were designated at the
genus level. As the probes are designed against conserved sequences, the
ViroChip can be used to identify new viruses within the same family as a
familiar, sequenced virus.

Advantages of virochip

ViroChip platform are the low cost of spotted oligo arrays and it can
detect novel viruses within a known family.

 

Disadvantages of
virochip

Ihe disadvantages of virochip can result mostly from the limitations of
spotted oligo technology.The low density of probes can be spotted on each
slide.It is an expensive
technology. The latest version of the ViroChip solves these issues following
the use of the Agilent ink-jet array . Probe designs have to be updated
occasionally to remain in progress with novel genomes in GenBank.

 

GreeneChip for pathogen detection

The ‘GreeneChip’
arrays show a broader-spectrum approach to pathogen identification .These chips
are high-density oligonucleotide arrays that are fabricated by using the
Agilent inkjet system. GreeneChipVr version 1.0 has 9477 probes for virus
infecting vertebrate. GreeneChipPm v1.0 is a panmicrobial array design that
contain all of  GreeneChipVr probes along
with probes for several thousand pathogenic bacteria, protozoa and fungi thus
containing 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 is
sensitive technique for virus identification.

DNA Microchip For The Diagnosis Of INFECTIOUS
DISEASES

·        
Infectitious
diseases by different species

Infectitious diseases are very serious
and human beings are continuously fighting to avoid pathogens. Due to new
pathogen emergence many infectitious diseases are spreading by viruses like immune deficiency syndrome
(AIDS), by bacteria like avian
influenza, malaria, measles and also by antibiotic
resistance superbacteria like methicillin-resistant Staphylococcus
aureus  vancomycin resistanct S. aureus.

·        
Reasons
of difficulty in identification of pathogens

Due to abuse of antibiotics, overuse of
the immunosuppressants in transplantation and overdose of drugs as anticancer
therapy it is getting impossible for the rapid detection and identification of
the pathogenic agents.

·        
Different
methods for identification of pathogen

Although various methods have made like
nucleic acid assay (e.g. PCR based assay), culture based assay ( in which
antibodies are being administrated and serological and immunological methods
but these methods has some limitations like low speed intensive labor and
resulting in high fast false results.

·        
Reason
why Microchip technology prevailed

Whereas, now Microchip technology has
been developed and successfully detected the microbial pathogens. It provides
identification of pathogens with increased assay capabilities like high
sensititivity specificity, high throughput detection thus disease is now
possible to be identified with greater ease and fastly so that better decisions
can be made.

Detection of Pathogenic Viruses by Microchip
technology

A virus is small infectious agentthat
invades living cell to replicate. Viral infection causes many symptoms like
sore throat, pain in body, common cold. Viral infection can be very dangerous
to the community because disease caused by viruses undergo virals and fast
contagiousness.

Identification of different virus using different
assay

It is necessary to get protection again
viral diseases faster.Viruses contain specific nucleic acid sequences that are 30% different in the same species or
even several types of serotypes exits. So ,it is not very difficult to detect
pathogens by nucleic acid-assays.

Hepatitis
virus identification by Quantitative nano gold-silver stain Microchip

Hepatitis virus cause chronic liver
disease and hepatocellular carcinoma. It is being detected from a patient’s
serum by quantitative nano gold-silver stain microchip.

 The use of gold nanoparticles treated with silver (AgNO3) for signal
amplification has increased sensitivity, thus allowing detection of viruse upto
100fM of ampicon.

 

Detection of the Human papilloma virus

Human papilloma virus (HPV) has almost
120 different types  which infects
through the skin and mucous membranes, also causes cervical cancer.

HPV detection chip

Researchers reported several specific
types of probes for HPV detection.Yoon and his coworkers developed microchip
for the detection of the HPV known as HPV detection chip then it was further
modified by Moon and his coworkers this Microchip having dual probes for the identification of 40 types of HPV. This assay
provides 100% sensitivity and 98% of the specificity relatively much better
results are obtained.

Probes were designed on the basis of the
sequence of the L1, E6 and E7 genes of almost 35 types of HPV virus thus are
complementarity to the sequence of probe.

Result

35 types of genotypes of HPV were
obtained from cervical cell species of 4898 Korean adult women and 68 cervical
cancer cases. Thus different disease can be diagnose and  identified with greater ease than the
conventional methods.

.

Identification of the Genetic diseases by Microchip
Technology

Genetic disorders are known to be as
hereditary disease due to inherited gene that causes abnormalities or mutations
and ultimately alters the functions of the normal gene and passes to next
generations resulting that particular disease in new born.

Classification of
genetic disorders

 Genetic disorders are classified into

      
I.           
Chromosomal  abnormalities

   
II.           
Mutations
in DNA sequence

1. Chromosomal Abnormalities Detection by
Microchip

“Genetic disorders that cause
chromosomal abnormality by Duplication,
Deletion, Translocation or Inversion of chromosomal part and results in
abnormality of chromosomes.”

Several different diseases are caused
due to chromosomal abnormalities like

·        
Down’s syndrome ( with
chromosome 21)

·        
Edwards syndrome (with
chromosome 18)

·        
Patau syndrome (with
chromosome 13)

·        
Turner syndrome (with
chromosome XO)

·        
Klinefelter syndrome
with having chromosome (XXY)

Microchip for the identification of  diseases caused by chromosomal abnormalities
and diagnosis

For all the chromosomal abnormalities
which causes diseases can be identified by the fabrication of the microchip
comprising specific chromosome related
BAC chip which plays important role in diagnose of abnormalities detected
and  it can be  reconfirmed by a method known as  fluorescence
in situ hybridization (FISH).

 

 

 

Fluorescence
in situ hybridization (FISH)

“FISH is a
method by which the position of specific DNA sequence present on chromosomes
can be located. Most of the situ
hybridization methods use fluorescent
probes for the identification of specific DNA sequence.”

Ø 
Successful in situ hybridization method based on
the stability of the DNA double
helix.

 

 

Principles of fluorescence in situ hybridization (FISH)

 

(a)     
Important elements
need to perform fish method is DNA probe and a target sequence.

 

(b)     
 DNA probe I labelled by methods random
primer 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 are
denatured at specific temperatures.

 

(e)      
By the combination of the denatured probe and specific
target sequence annealing of the complementary DNA sequence is done.

Ø  In indirect labeling of the probe an
extra step is to be done for the visualization of the hepten that are not fluorescent and thus can be use in the
different immunological detection systems.

 

Advantages
of  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

“Mutation
in 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 that
cause different disease phenotypes.”

Identification of mutation and diagnosis by
microchip

Genetic
counselling is the
method in which entire genome of the patients or relatives having risk of
inherited disorders is sequenced to know about the nature, probability,
developing and transmitting of disease so that important decisions like type of
treatment or in family planning can be made

 

 

Ø  Microchips
are used for diagnosis and identification of many diseases using classical
method known as genetic counselling,

SNPs detection

Microchip plays an important role in
identification of diseased genes, particular those genes causing different
mutations in stages of life.

Example                                                                                                                    

Ø  Cardiovascular
disease id diagnose by the identification of multiple single nucleotide known as polymorphisms
in disease-relevant genes, results in the different patterns of genotypes.

 

Detection of SNPs in Four Steps By Microchip

1.      Hybridization

2.      Cleavage
of present mismatch sites

3.      To
denture the  hybridized Dimer

4.      Detection
of four different  probes with dsDNA

Identification of Mutations Caused by  Monogenic Disorders

Monogenic
Disorders are also known as Single Abnormal Gene and there is a single
defective gene present on autosomes.

·        
Autosomal
dominant diseases

Avellino
Corneal dystrophy is due to mutation occurring in gene that is coding for
?IG-H3 which is autosomal dominant diseases
can be homozygous mutation and heterozygous mutation. It starts with blurry
symptom in centre of the cornea and ends up with vision loss with aging.

Method of identification of disease by using
Microchip

Microchip for the cure of the corneal
dystrophy can be used for detecting the mutations in Exons 4 and 12 of the
?IG-H3.

 

Conditions
provided to Microchip

Ø  Microchip
was optimized at very high specificity and sensitivity

Ø  Captured
probe concentration should be at range of 
(100, 50, 30, and 10 µM),

Ø  The
length of probe should be of  11 to 17
polymers

Ø  Hybridization
time given should be (1, 2, 3, 4, 6, and 24 hrs.).

Method

 

Result

Cheugu
invented a complementary DNA microchip
that allows identification of different genetic mutation present in the
heterozygous mutation carriers and normal patient.

Above performed chip method used blood
samples of 98 patients that give excellent result with 100% of accuracy.

Cancer identification
by microchip

Microchips initially used by Martin and
his colleagues for the identification of the 170 gene expressed differently in
the present of breast tumour cells.

Then
different rarer cancer cells like

·        
Skin cancer cells,

·        
 salivary gland cancer,

·        
 sarcoma cancerous cells ,

·        
 small cell lung cancer

·        
 small intestine cancer cells were
identified by the use of microchip

 

 

Method of identification of rare cancer cells

Future prospective
regarding to the Microchip in disease identification

·        
Modifications

For its increase in the practical use of
the Microchip it is being making affordable, convenient to practice, accurate
and portable..

Example

Such
lateral flow assay should provide diagnosis of many diseases in clinical and
environment sample by on site pathogen and SNP detection

·        
Detection
of chromatin

By combining of microchip assay with
different kinds of other techniques it is making possible to detect even
chromatin level changes.

·        
Genome
projects

Many genome projects and infectious
organisms are providing large amount of information about genome sequencing and
thus by using probes many different diseases at gene level are making possible
to identify and diagnose easily.

·        
Smart
drugs

With advancement in microchip technology
multiple genetic markers and their functions will be identified in short future
and then this information will be used by medical researchers to produce and
diagnose patients with smart drugs.

 

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