“The SNP array or SNP microarray works on the principle of DNA hybridization in which a single base change can be detected through fluorescence chemistry.”
SNP or snip is known as single nucleotide polymorphism. Simply put, a change, alteration or polymorphism in a single base is known as SNP or single nucleotide polymorphism.
Our genome is made up of 3.2 billion basepairs distributed on 23 chromosomes. Through replication, genetic material replicates from one cell to another. During replication, the polymerase inserts nucleotides, but sometimes it may incorporate mismatch or wrong nucleotide that makes a change at a single base.
However, not only replication but also other extrinsic factors are causing SNPs.
On average, an SNP occurs after every 1000 bases in our genome. A change is considered as SNP if its frequency is more than 1% in a genome.
Key Topics:
What is an SNP array?
SNPs may be associated with complex traits and multigenic disorders. PCR can only detect a few SNP at once therefore a microarray-based SNPs array is developed in order to screen SNPs from a genome.
The SNP microarray or SNP array has significant importance to know disease susceptibilities. And to known single-base variations associated with a complex trait.
The method is also used in drug studies, linkage analysis, population-based genotyping studies, gene mapping, and marker assistant selection studies.
The SNP is a kind of marker involved in many disorders and conditions thus it can be used to study the effect and frequency of SNP in a population and between two populations.
Technically, it is a hybridization-based technique that relies on the principle of fluorescence.
Related article: Genome-On-A-Chip: DNA Microarray.
How does SNP array work?
Like any other microarray methods, SNP array completes in three common steps:
- Immobilizing oligonucleotides (making a chip)
- Fragmenting and labeling nucleic acid
- Hybridization
Making an array chip:
The array chip is a glass slide on which sequence-specific oligo probes or allele-specific oligonucleotides (ASO) are immobilized. The SNP array is based on the sequence information, a common SNP based microarray chip is manufactured by chemical spray method, micromechanical printing method or by in situ synthesis method.
On a small piece of slide around 1cm, approximately 4,00,000 different sequence-specific probes are immobilized. (It can’t see naked eye). Various array chips based on different populations and sizes of common SNPs is now commercially available.
Fragmentation and labeling of nucleic acid:
To perform an SNP array we need pure nucleic acid or DNA and to do so, we have to first isolated DNA from a sample. You can use any DNA extraction method but make sure it can give you highly pure and high yield DNA.
I recommended reading this article before doing DNA extraction: 10 Secret tips to isolated DNA from a sample.
Check the purity and quantity of a nucleic acid before doing hybridization. Now in the next step, we have to fragment our gDNA.
Enzymatic digestion or physical fragmentation methods can be practiced which makes small fragments of our gDNA. The whole gDNA is complex to process henceforth we need to fragment it.
By PCR amplification DNA fragments are amplified until a sufficient amount of DNA fragments are obtained. In the next step, the DNA fragments are labeled with a fluorescent dye. Different fluorescent dyes are handled by different manufacturers. Once DNA is labeled, unlabeled DNA is washed off.
Hybridization:
Hybridization between the oligo probes and nucleic acid occurs on a glass slide on which we have immobilized probes.
To facilitate hybridization between DNA fragments and probes several conditions are followed. Use a buffer or reaction buffer to increase the hybridization efficiency (that must be provided by a manufacturer).
Wash of unbounded nucleic acid fragments using washing buffer several times so that it can’t make hurdles in final results. Allow the hybridization at 37°C for at least 24 hours.
A microarray scanner scans the entire slide to detect hybridization. Once the scanning is done, the results are sent to the computer where a software analyses the results.
At the hybridization site, the nucleic acid fragments bind to the probe only when it finds its exactly complementary site. And once it hybridizes, a fluorescent molecule releases fluorescence.
A detector detects the amount of fluorescence emits by hybridization and gives a signal as true hybridization. From every single probe immobilized our of 1,00,000 to 5,00,000 signals are generated, collected, and process to get results.
Results of SNP microarray:
In the SNP microarray, we have designed a slide or array based on the information of single-nucleotide polymorphism of a population or associated with a disease or group of diseases.
Therefore, a particular SNP is present in a sample (which we wish to study and made a probe specific to it), it binds or hybridizes. And as we said, the fluorescence signals are recorded.
Based on prior information on the SNP database, a graph is made. It looks like this,
Anyway, the results analysis part is not our cup of tea, a bioinformatics expert can do it only. But I can give you an overview. First, arrange our SNP database as per the chromosome and located every SNP on a chromosome. It will look like this.
Now in the next step, although I am neither bioinformatics expert nor statistic experts but can say what happened.
Every SNPs log10p value is calculated if it is above 4.6 it is considered as suspected SNP, associated with a disease or phenotype, less than 4.6 do not consider and discarded, otherwise.
By doing in-depth bioinformatics studies we can locate SNP on a gene, its function, what alteration it may cause, and other related information.
Applications of SNP array:
As we said, the SNP marker is now a day widely studied to link various complex and multifactorial disorders. Besides, it is used in
- Haplotype and gene mapping
- Cancer research
- Personalized genetic analysis
- Genetic medicine research
- Genome-wide association studies
Conclusion:
SNP microarray is a bit complicated process and involves extensive bioinformatics in it. You might think that how we can prepare a chip in our lab? But let me tell you, don’t worry about it. Ready to use SNP chips are now available in the market.
We only have to perform microarray hybridization. Even, we can send results to other bioinformatics labs to evaluate SNPs results.
The present method is time-consuming and very costlier for now at least. Because companies have to invest huge money to develop a chip and scanners. Furthermore, prior sequence information is must required to develop as well as evaluate SNP chip and chip results.
