2.2.3: SNPs and microarrays

What is a SNP?

A single-nucleotide polymorphism, or SNP, is a molecular marker consisting of a single base-pair difference between two individuals at the same location in the genome. For example, one allele may have an A at a particular location, while another allele has a C. Single base insertions and deletions are also SNPs. When SNPs occur in protein-coding genes, they are responsible for point mutations such as missense, nonsense and frameshift mutations. However, the majority of SNPs in humans occur outside of a gene, and most often have no discernable phenotypic impact. In humans, SNPs are very common: one estimate suggests that there is a SNP (with a population-wide frequency of at least 1%) on average every 300 bases. That is to say, with an approximate genome size of 3 billion basepairs, there are about 10 million SNPs in the human genome!

Because they are so common, SNPs are extraordinarily useful in genetics research. Later we will explore their use in gene mapping and association studies; for now, let's consider how one can determine the genotype of tens of thousands of SNPs at once using DNA hybridization and a technology called a DNA microarray.

DNA Microarrays

A DNA microarray (also commonly known as a DNA chip or biochip) is a collection of microscopic DNA spots attached to a solid surface. Each DNA spot contains picomoles (10⁻¹² moles) of a specific DNA sequence, known as probes (or reporters or oligos). These can be a short section of a gene or other DNA element that is used to hybridize a cDNA or cRNA (also called anti-sense RNA) sample (called target) under high-stringency conditions. Probe-target hybridization is usually detected and quantified by the detection of fluorophore-, silver-, or chemiluminescence-labeled targets to determine the relative abundance of nucleic acid sequences in the target.

The core principle behind microarrays is a hybridization between two DNA strands, the property of complementary nucleic acid sequences to specifically pair with each other by forming hydrogen bonds between complementary nucleotide base pairs. A high number of complementary base pairs in a nucleotide sequence means tighter non-covalent bonding between the two strands. After washing off non-specific bonding sequences, only strongly paired strands will remain hybridized. The total strength of the signal from a spot (feature) depends upon the amount of target sample binding to the probes present on that spot.  Figure \(\PageIndex{2}\)s shows the hybridization of target and probe DNA on a microarray.

Figure \(\PageIndex{2}\): Hybridization of Target DNA with the Probe DNA During Microarray Analysis. Image by Squidonius

Many types of arrays exist and the broadest distinction is whether they are spatially arranged on a surface or coded beads:

• The traditional solid-phase array is a collection of orderly microscopic "spots", called features, each with thousands of identical and specific probes attached to a solid surface, such as glass, plastic, or silicon biochip (commonly known as a genome chip, DNA chip or gene array). Thousands of these features can be placed in known locations on a single DNA microarray.
• The alternative bead array is a collection of microscopic polystyrene beads, each with a specific probe and a ratio of two or more dyes, which do not interfere with the fluorescent dyes used on the target sequence.

 

How do you use a DNA microarray to genotype a SNP?

Simple! For each SNP you want to detect, you design one probe per SNP allele. For example, imagine you are designing the probes on your microarray to detect the genotype of a SNP that can be an A or a C. Assume the probes on your SNP chip are 30 bp long.

• First, design one probe with the 30 bases surrounding the SNP and an A at the polymorphic base
• Next, design a probe that is identical except for changing the polymorphic A to a C.
• Break the DNA you're analyzing into very short pieces, label it with a green fluorescent dye and hybridize it to the chip.

If the "spot" on the chip with the A is brigher than the spot with the C, that means that the DNA bound more tightly to the spot with the A -- and the genotype of the individual is homozygous A at that SNP. Conversely, if the spot with the C is brighter than the spot with the A, the genotype of the individual is homozygous C.

Note

What would it mean if the spot on the chip with the A was the same brightness as the spot on the chip with the C?

Because microfabrication technology allows us to make very small spots on glass or silicon, modern DNA microarrays can have hundreds of thousands of probes, genotyping tens of thousands of alleles at once. And because they are produced at scale, genotyping all these SNPs costs less than a hundred dollars per sample.

 

Note

Microarrays are also commonly used to measure gene expression. If instead of having the probes be sequences of alleles, you made them with sequences of messenger RNAs, then you can use the following method to measure gene expression:

• Extract all the RNA from the cells you're interested in
• Use a reverse transcriptase enzyme to convert them into cDNA
• Label the cDNA and hybridize it to your gene expression chip
• Measure the amount of fluorescence at each probe

The brighter the probe is glowing, the more mRNA was in the original sample, and the more that gene was being expressed.