“The DNA microarray is a molecular genetic technique uses nucleic acid hybridization principle for gene expression studies, identification of genotype and mutations associated with the disease.”
After the discovery of the PCR by Kary Mullis the concept of DNA microarray was developed by Edward in the late ’90s.
The method was discovered in 1988 by Edward, Mark Schena was the first scientist to use it for gene expression studies in 1995. The first human transcriptome stud is published in 1996.
Before the discovery of DNA microarray, the PCR is used for genotyping, identification of single-gene mutations and quantification of gene expression.
However, the PCR has one major limitation, it can study only a limited number of mutations or gene expression.
Hence the need for an instrument capable of studying thousands of mutations, estimating the gene expression of the entire transcriptome is arise.
And this need established the base for the development of a microarray platform which is actually inspired from the southern blotting, not from the PCR.
Unlike the temperature-depended PCR amplification, the chemistry of microarray is based on the probe hybridization.
Read more on PCR: The polymerase chain reaction.
The present article is all about DNA microarray and its application, the content of the article is:
What is DNA microarray?
Read first: A Brief Introduction To Cytogenetics [Karyotyping, FISH and Microarray].
“A DNA microarray is a technique used to detects thousands of mutations and related gene expression at once.”
The DNA microarray is also known as a DNA chip, gene chip, biochip or whole-genome microarray.
[epcl_box type=”information”]Array: An arrangement of something in an orderly manner.[/epcl_box]
The probe chip, sample DNA or RNA and microarray detector are three major elements need for microarray.
Principle of DNA microarray:
The principle behind the DNA microarray is based on the nucleic acid hybridization. A fluorophore bind to the DNA strand emits fluorescence once it hybridize with the nucleic acid probe.
Millions of short and single-stranded DNA probe are placed on a glass slide or solid matrix. Adding the fragmented DNA sample allows hydrogen bonding between the probe and the target DNA strand if both are complementary to each other.
The fragmented DNA is labelled with the fluorophore thus when it hybridize, it emits the fluorescent detected by the microarray detector.
The idea behinds the DNA microarray has come from the concept of southern hybridization method.
However, the method is a bit different, in southern hybridization, instead of the probe, the target DNA is placed or immobilized on a nylon paper and allows hybridization by adding probes.
Only a few probes can be hybridized in southern blots, however, in the microarray, instead of target DNA, probes are immobilized on a glass slide, which allows many probes to hybridize at multiple complementary sites.
One of the major application of DNA microarray is that it has the power to study the whole transcriptome.
Thes set of all mRNA of a cell, expressed at any given time is called the transcriptome.
Designing a microarray chip:
The microarray chip is a solid glass, silicon or nylon surface on which thousands of probes are spotted.
Using the surface engineering method, the spots of probes for different DNA sequences are immobilized on a glass surface using fine needles or a 3D printer.
Process of DNA microarray:
Isolation of nucleic acid:
The process of microarray starts with RNA isolation.
Here, we want to study the gene expression, therefore, we are extracting RNA and uses the mRNA only for microarray.
The total RNA contains all types of tRNA, rRNA, mRNA and other smaller RNAs which obviously we do not want to study.
The mRNA is a gene product that expressed into protein, thus we will only extract the total mRNA by using the poly- T-tail containing column beads.
As we know, that the mRNA has a poly-A tail, it binds with the poly-T of a column and all other RNAs washed off using the wash buffer.
The only mRNA remains in a column which is eluted using the elution buffer.
Reverse transcription and labelling of cDNA:
The mRNA is reverse transcribed into the cDNA using the reverse transcription PCR. The cDNA is then fragmented or digested with the restriction digestion.
After that, the sample DNA, as well as the control DNA, is labelled with the fluorophores. Two different labelling dye is used for two different samples.
The cDNA from the sample as well as the control is labelled with Cyt3 blue and Cyt5 red dye, respectively.
Hybridization:
Now, the labelled DNA fragments are applied on microarray slides which already have the know DNA probes.
The sample and the control DNA competes with each other for hybridization, if it finds its complementary sequence it will bind.
Otherwise, the unbounded DNA is washed off during the post hybridization wash.
Scanning and data collection:
Once the DNA finds its complementary sequence it hybridize with it and emits fluorescence. The fluorescent signals are collected by the microarray scanner.
A camera in it captures the signal and send it to the computer. The computer software analysed the data.
The amount of fluorescent emitted through the hybridization is proportional to the amount of the target DNA binds to the probe.
“More hybridization, more intense signal, more amount of gene expression.”
Based on the data collected from each spot, the computer software analyses the data and quantify the gene expression.
This is the brief outline of the DNA microarray procedure, especially, for the gene expression assay. Many variations of DNA microarray are now available in the market.
For example the SNP microarray.
The SNP microarray/ SNP array/ whole chromosome SNP microarray or whole chromosome microarray method estimate the copy number variations instead of gene expression.
In the typical PCR based genotyping method a single or several SNPs can be identified but in the SNP microarray, millions of different SNPs and copy number variation are characterised.
Although the mechanism of SNP microarray is same as the conventional microarray. But here instead of RNA, DNA is used as starting material.
The SNP specific probes are immobilised on the glass or solid surface.
The DNA is extracted from the sample and fragmented using restriction digestion, simultaneously send to the microarray lab.
The DNA of the patient as well as the normal sample is applied to the microarray for hybridization after fluorescent labelling.
The rest of the procedures are the same but instead of the gene expression, the final results indicate the presence or absence of SNP in a sample.
Millions of different SNPs of different chromosomes are being screened using the SNP analysis and thus it is one of the best tools for the population study and estimating genotype frequency.
Moreover, it is now one of the best available tool detection of the predisposition of the disease.
Several applications of Whole chromosome SNP microarray are:
- Identification of SNPs
- Detection of copy number variations
- Linkage analysis
- Genotyping study
- Population study
- Forensic studies
- Predisposition and disease diagnosis
Related article: An Introduction To Single Nucleotide Polymorphism (SNP).
Results of microarray:
Now in this section, I am giving you a brief outline of the microarray results.
See the image carefully, these are the results of a typical whole chromosome microarray. As you can see, the blue spots are our patient’s DNA hybridized with probes on a particular location of a chromosome.
Whereas red spots are our control DNA.
Now it is impossible to interpret the results by naked eyes, a computer software analysed the results based on the signals of hybridization.
But I want to show you one this.
Our sample is female having two XX chromosome, therefore you can observe no hybridization of patient’s DNA (no blue spots) only red spots are present which indicates the hybridization of the control sample.
On the other side, on the X chromosome, our sample is hybridized more in comparison with the control DNA, more blue spots are observed (due to the presence of two X chromosome).
The lacks or blanks in the result (chromosome 1 and 9) indicates no hybridization.
Applications of DNA microarray:
Obviously the first and foremost application of DNA microarray is the study of transcriptomics.
The transcriptome is a complete set of total mRNA of a genome going to translate into protein.
The gene has the sequences to form the protein which intermediately transcribed into mRNA and then translated into the protein.
By estimating the mRNA formed during transcription, we can estimate in which amount the protein forms.
Therefore, we can even study proteomes using microarray.
Disease diagnosis:
In modern days, one of the fascinating application of microarray is in the diagnosis of many inherited as well as non-inherited disease.
Multigenic disorders can not be studies during the PCR method, the microarray can analyse so many genes at a time, therefore, mutations in a gene or mutations of many genes can be studied and analysed using the microarray.
Even the microarray is the first choice for the diagnosis of rare genetic conditions and predisposition of cancer.
Gene expression between two tissues (tissue-specific transcriptome study), is possible using DNA microarray.
For that,
mRNA from two different tissues are extracted, reverse transcribed into cDNA and labelled with two different fluorescent dye.
Both tissue-specific cDNA complete with each other for hybridize with probe settled on a glass slide.
The intensity of the signal emitted is directly proportional to the amount of hybridization and hence the expressions of different genes in different tissues can be determined.
It is used in the genomic gain and loss studies.
As we discussed above, in the SNP microarray, all the copy number variation from different chromosomes can be determined, thus we can measure the amount of sequence deleted or loss and add into the genome.
In another type of DNA microarray, different microorganism present in different environmental DNA can be identified in a single microarray experiment.
New mutations associated with the disease can be characterised.
The microarray technique is based on the information of mutation available with us, unlike the DNA sequence, it can not determine new mutations or variations.
But it can be used for studying the association of mutations with the disease we wish to study, thus genome-wide association study.
In addition to this several other application of DNA microarray are:
- GMO plant studies
- Population study
- Gene and genotyping studies
- Forensic studies
- Pharmacogenomic studies
- Cancer research
Limitations of DNA microarray:
Costlier,
As we know that the microarray analyses the entire genome, it is obvious that the cost of the array will be more.
Though the method is accurate, it is time-consuming.
A lot of time required to analyse and interpret the results even though it is fully automated.
It is non-reproducible.
It can estimate the relative concentration in the case of gene expression, not absolute quantification.
It can only detect known mutations or gene which are known.
Conclusion:
After the DNA sequencing, the DNA microarray is one of the advanced method available in genetics and genomics studies.
In spite of costlier, it has the ability to analyse thousands of queries in one assay and that actually makes it powerfull above all methods available.
Sources:
- Bumgarner R. Overview of DNA microarrays: types, applications, and their future. Curr Protoc Mol Biol. 2013; Chapter 22:Unit–22.1.
- Govindarajan R, Duraiyan J, Kaliyappan K, Palanisamy M. Microarray and its applications. J Pharm Bioallied Sci. 2012;4(Suppl 2): S310–S312.
