If a microbiologist is studying bacteria that premeditate, or break down, toxic wastes and wants to know which specific genes are active when that bacterium is degrading, say, PCBs, he would likely use a tool called the DNA microarray. Microarrays enable scientists to monitor the activities of hundreds or thousands of genes at once. All microarrays (also called DNA chips or gene chips) work on the basic principle that complementary nucleotide sequences in DNA (and RNA) match up like the two halves of a piece of Velcro coming together. Pattern of gene activity on a microarray chip. A microarray consists of an orderly arrangement of bits of genetic material in super-tiny spots laid down in a grid on a suitable surface, often a glass slide with a specially chemically treated surface.

1. 1
DNA MICROARRAY
(DNA CHIP)

2. Microarray
 Introduction
 principle
 Applications
 Advantages
 Limitations
 Conclusion
 References

3. Introduction:
Definition: DNA microarrays are solid supports, usually of glass or
silicon, upon which DNA is attached in an organized grid fashion.
Each spot of DNA, called a probe, represents a single gene.
There are several synonyms of DNA microarrays such as DNA
chips, gene chips, DNA arrays, gene arrays and biochips.
[Ref: Presscot (Book for Microbiology), www.wikipedia.org]
History: Microarray technology evolved from Southern blotting
The concept of microarrays was first proposed in the late 1980s by
Augenlicht and his colleagues.
The use of miniaturized microarrays for gene expression profiling was
first reported in 1995, and a complete eukaryotic genome
(Saccharomyces cerevisiae) on a microarray was published in 1997 by
Pat Brown’s group

4. a) A DNA chip can be
manufactured to contain
hundreds of thousands of
synthetic single-stranded DNA
sequences.
b) Unknown DNA from a patient is
separated into single strands,
enzymatically cut and labeled with
a fluorescent dye.

5. c) The unknown DNA is inserted into the
chip and allowed to hybridize with the
DNA on the chip.
d) The tagged DNA will bind only to the
complementary DNA on the chip. The
bound DNA will be detected by its
fluorescent dye and analyzed by a
computer. The red light is a gene
expressed in normal cells; green is a
mutated gene expressed in tumor cells;
and yellow, in both cells.
Fig: DNA Chip Technology

6. Principle
The principle of DNA microarrays lies on the hybridization
between the nucleotide. Using this technology the presence of
one genomic or cDNA sequence in 1,00,000 or more sequences
can be screened in a single hybridization.
The property of complementary nucleic acid sequences is to
specifically pair with each other by forming hydrogen bonds
between complementary nucleotide base pairs.

7. – By using an array containing many DNA samples, scientists can
determine, in a single experiment, the expression levels of
hundreds or thousands of genes within a cell by measuring the
amount of mRNA bound to each site on the array.

8. Table 1. Steps in the design and implementation of a DNA microarray
1) Probe (cDNA/oligo
with known identity)
2) Chip
fabrication
(Putting
probes on the
chip)
3) Target
(fluorecently
labeled
sample)
4) Assay 5) Readout 6) Informatics
Small
oligonucleotides,
cDNAs,
chromosome.
Photolithogra
--phy, pipette,
piezoelectric.
RNA,
(mRNA)
cDNA.
Hybridization.
Fluorescenc
e, probeless
(conductanc
e, MS,
electrophore
sis).
Robotics
control,
Image
processing,
DBMS,bioinf
ormatics.

9. 1. Sample
preparation
2. Purification

10. 10
3. Reverse
Transcription
4. Labelling

11. 5.Hibridization
6. Scanning
7.Normalization
and analysis

12. There are 2 types of DNA Chips/Microarrays:
Types of DNA chips
1. cDNA based microarray
2. Oligonucleotide based
microaaray

13. Applications
Gene expression analysis
– Not all the genes in the human genome are active at all times
– used to detect DNA , or detect RNA that may or may not be translated into
proteins.
– The process of measuring gene expression via cDNA is called expression
analysis

14. – Thousand genes are simultaneously assessed
– Study the effects of certain treatments, diseases, and developmental stages on
gene expression.
– E.g.: identify genes expression changes due to pathogens or other organisms
by comparing with uninfected cells or tissues

15. Help to investigate about different diseases
– E.g.: Earlier cancers classified on the basis of the organs in which the tumors
develop.
– Now, classify the types of cancer on the basis of the patterns of gene activity in
the tumor cells.
– Help to produce very effective drugs
Disease diagnosis

16. Extensive application in Pharmacogenomics
– Comparative analysis of the genes
– Help the identification of the specific proteins produce by diseased cells
– Information used to synthesize drugs which combat with these proteins and
reduce their effect.
Drug Discovery

17. Toxicological Research
– A rapid platform for the research of the impact of
toxins on the cells and their passing on to the
progeny.
– Important for Toxicogenomic studies

18. – Small microarrays to check IDs of organisms in
food and feed (like GMO) and mycoplasmas in
cell culture
– Mostly combining PCR and microarray
technology
Gene ID

19. Nutrigenomic research
Study variations in the genes related to the influence of diets.
– These variations, known as single nucleotide polymorphism

20. – E.g.: Studies are followed to reveal,
– Effects of calorie restriction on gene expression
– Obesity and high-fat diets
– Genes responds to gluten and soy protein

21. Microarray
application in
microbiology

26. Microbial Diagnostic Microarray
– specific oligonucleotides, 35-70 nucleotides, in length are
chemically synthesized
– the oligonucleotides are spotted, at defined positions, onto a
glass slide to construct the array
– binding of fluorescently labelled probes to the oligonucleotides
is detected spectrophotometrically

27. Genes USED ON MDM?
– Housekeeping genes
– 16S rRNA,
– 16S-23S rRNA intergenic transcribed spacer region (ITS),
– rpoB; RNA polymerase Beta
– Hsp60/groEL; Heat shock protein
– recA; Recombinase A
– gyrB; Gyrase Beta

28. CONT.
– Virulence genes
– bacterial toxins
– adhesins
– important virulence factors
– facilitate colonisation
– cause tissue damage

29. BACTERIA DETECTION

30. – The complex interaction between a pathogen and its host is the molecular basis
of infectious diseases. Microarray technology is a powerful tool to investigate
the crosstalk between a pathogen and the host as it assesses whole genome
expression profiles in response to disease

31. 1. Tuberculosis
– Gryadunov et al : developed a biochip for detection of rifampicin-resistant and
isoniazid-resistant strains of M. tuberculosis .
– The biochip identifies over 95% of rifampicin-resistant and more than 80% of
isoniazid-resistant M. tuberculosis strains in sputum samples.
– The biochip has 77 gel elements and detects the 27 most-common mutations in
the rpoB gene responsible for rifampicin resistance as well as 11 mutations in
the katG gene, five mutations in the promoter region of the inhA gene, and five
mutations in the intergenic regulatory region of the ahpC-oxyR genes all of
which can cause resistance to isoniazid.

32. Cont.
– sensitivity of 80% and a specificity of 100% compare to traditional testing for
rifampicin resistance.
– Disadvantage: rare mutations or unknown mutations not detectable by the
microarray probes
The newest generation of TB-biochips identifies mutations responsible for the
emerging resistance of M. tuberculosis so the highly effective second-line
fluoroquinolone antibiotics can be administered

33. 2. Meningitis
– Using the sequencing array, the scientists were able to correctly classify 45
samples that were previously identified by conventional methods. But more
importantly, was able to classify 12 previously unclassifiable samples into
existing meningitis serotypes.
– resequencing microarrays provide results in just 48 hours, much faster than
traditional methods.
– The meningitis resequencing array can now be used to quickly identify new
meningitis strains, as well as for epidemiological studies and vaccine research.

34. PARASITE DETECTION

35. VIRUS DETECTION
– Based on publications, microarray approaches can be summarized into four viral
infection groups to focus on:
1) respiratory diseases
2) hemorrhagic fever (HF)
3) neurotropic infection
4) HIV

36. 1. Respiratory Diseases
Detection of etiological agents :
– Some influenza microarrays are designed to detect DNA. For instance, a
universal microchip was developed for genotyping influenza A viruses with two
sets of oligonucleotide probes allowing viruses to be classified by the subtype of
hemagglutinin (H1 - H13, H15, H16) and neuraminidase (N1 - N9)

37. 2. Hemorrhagic Fever
– Viruses associated with hemorrhagic fever (HF) are mainly found in the families
Arenaviridae,
– Bunyaviridae,
– Flaviviridae
– Filoviridae.

38. – Based on microarrays, a detection and identification approach was designed for
seven agents of the Flaviviridae family:
– yellow fever,
– West Nile virus (WNV),
– Japanese encephalitis,
– and the dengue 1 - 4 viruses,
which are causing severe human disease in tropical and subtropical areas all over
the world

39. 3.Neurotropic virus
– A DNA microarray for the detection of 13 specific pathogens in meningitis and
encephalitis cases was developed for the most common neurotropic viruses
including HSV-1, varicella-zoster virus [VZV], and enteroviruses.
– Also, a microarray comprising of 38 gene targets was developed for the
detection of several other viruses capable of causing CNS syndromes.

40. cont.
– This concluded that clinical sensitivity,specificity, and negative and positive
predictive values of the assay were 93%, 100%, 100%, and 83%
respectively,comparing microarray to the single-virus PCR

41. 4.HIV
– detect the pathogen but also can measure the amount of virus, a microarray
was developed by combining both methodologies. The study described an
original approach for simultaneous quantitative identification of these viruses in
blood plasma specimens using real-time PCR with primers immobilized on a
microarray

42. Another novel use of microarrays
with HIV
– the identification of resistance biomarkers on HIV-1,including pathways that
may be critical in anti-HIV-1vaccine design.

43. FUNGI DETECTION
 Studies reported the use of microarrays to identify pathogenic yeasts and molds
by targeting the ITS regions in fungal rRNA genes
 In 2007, Birgit Spiess et al. reported a sensitive DNA microarray to detect and
identify DNA from 14 fungal pathogens: Aspergillus fumigatus, Aspergillus
flavus,Aspergillus terreus, Candida albicans, Candida dubliniensis, Candida
glabrata, Candida lusitaniae, Candida tropicalis, Fusariumoxy sporum, Fusarium
solani,Mucor racemosus, Rhizopus microsporus, Scedosporium prolificans, and
Trichosporon asahii in blood, bronchoalveolar lavage, and tissue samples from
high-risk patients. The results in clinical samples from neutropenic patients
showed the specific detection of the 14 fungal pathogens by using a
combination of multiplex PCR and consecutive DNA microarray hybridization.
The capture probes were derived from unique sequences of the 18S, 5.8S, and

44. • Provides data for thousands of genes.
• One experiment instead of many.
• Fast and easy to obtain results.
• Huge step closer to discovering cures for diseases and cancer.
• Different parts of DNA can be used to study gene expresion.
ADVANTAGES
[Ref: www.biotechnologyforums.com, www.ehow.com]

45. Disadvantages:
• The biggest disadvantage of DNA chips is that they are expensive
to create.
• The production of too many results at a time requires long time
for analysis, which is quite complex in nature.
• The DNA chips do not have very long shelf life, which proves to
be another major disadvantage of the technology.
•Correlations in results do not mean causation
•Very little knowledge is available about many genes
•Just because mRNA is "turned on" doesn't mean proteins are made
•The findings may lead to unethical medical procedures
•Scientists have no standardized way to share results
[Ref: www.biotechnologyforums.com, www.ehow.com]

46. DNA Microarrays are one of the most effective invention ever developed. A
DNA Microarray is a test that allows for the comparison of thousands of genes
at once. Microarray technology uses chips with attached DNA sequences as
probes for gene expression. Any DNA in the sample that is complementary to a
probe sequence will become bound to the chip. Microarray technology is most
powerful when it used on species with a sequenced genome. The microarray
chip can hold sequences from every gene in the entire genome and the
expression of every gene can be studied simultaneously. Gene expression data
can provide information on the function of previously uncharacterized genes.
Conclusion

47. Net Source:
• www.wikipedia.org
• www.gene-chips.com
• www.biotechnology4u.com
• www.biotechnologyforums.com
• www.ehow.com
•http://www.bio.davidson.edu/courses/genomics/chip/chip.html
•http://www.cs.washington.edu/homes/jbuhler/research/array
Reference