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DNA MICROARRAY(DNA CHIP)

Microarray

Introduction

principle

Applications

Advantages

Limitations

Conclusion

References

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 blottingThe 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

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.

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

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.

– 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.

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.

Fluorescence, probeless (conductance, MS, electrophoresis).

Robotics control, Image processing, DBMS,bioinformatics.

1. Sample preparation

2. Purification

103. Reverse

Transcription

4. Labelling

5.Hibridization

6. Scanning

7.Normalization and analysis

There are 2 types of DNA Chips/Microarrays:

Types of DNA chips

1. cDNA based microarray

2. Oligonucleotide based microaaray

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

– 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

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

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

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

– 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

Nutrigenomic research

Study variations in the genes related to the influence of diets.

– These variations, known as single nucleotide polymorphism

– 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

Microarray application in microbiology

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

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

CONT.

– Virulence genes

– bacterial toxins

– adhesins

– important virulence factors

– facilitate colonisation

– cause tissue damage

BACTERIA DETECTION

– 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

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.

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

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.

PARASITE DETECTION

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

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)

2. Hemorrhagic Fever

– Viruses associated with hemorrhagic fever (HF) are mainly found in the families Arenaviridae,

– Bunyaviridae,

– Flaviviridae

– Filoviridae.

– 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

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.

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

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

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.

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 internal transcribed spacer one region of the fungal rRNA genes which contribute significantly in improving the diagnosis of IFI

• 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]

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]

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

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