Special Column on Breeding | Solid Phase Chip for 4-Genotyping

Frontier sharing

2022-10-07


Previously introduced to
Gene/SNP typing is the key of modern molecular breeding
There are many techniques and methods for SNP detection
Such as gene chip, simplified genome sequencing, re sequencing, targeted sequencing, etc
A good SNP typing method should take into account factors such as flux, cost, stability and flexibility
Therefore, several common SNP typing methods are introduced in the small staffing plan
This article starts with gene chip.

 

Introduction to Gene Chip

The layman who hears "gene chip" for the first time can easily connect it with the electronic chip of industrial integrated circuit. In fact, except that they all use micro technology to make the appearance more similar, they have nothing to do with each other and are purely porcelain. Gene chip, also known as DNA chip, biochip or DNA microarray, is based on the principle of specific interaction between molecules, integrating discontinuous analysis processes on the surface of solid phase chips such as silicon or glass, to achieve accurate, rapid and large detection of cells, proteins, genes and other biological components. According to specific scientific research and application contents, gene chips can be subdivided into microarray comparative genomic hybridization (a-CGH) chips, microRNA chips, SNP chips, expression profile chips, DNA methylation chips and chromatin immunoprecipitation chips.

The first commercial gene chip was born in 1996. With the development of biotechnology, so far, biochips containing high-density molecular markers or probes have played a huge role in the field of biomolecular detection and genotype identification, and have been widely used in medical detection and diagnosis, genomics, genetics and animal and plant breeding. In particular, a large amount of chip data has been generated in the medical research field. The GEO of NCBI is the largest chip database at present, and has also become an important source of public data mining for many researchers. Compared with traditional detection methods, gene chip can fix tens of thousands of probes at one time to detect the corresponding information of tens of thousands of known sequences.

SNP chip technology is to fix the third generation SNP marker on the carrier to form a dense array of oligonucleotide probes, and then conduct allele specific reaction with the target DNA. The SNP site polymorphism is determined according to the presence and strength of the signal after the reaction. This technology can realize rapid and high-density scanning of the whole crop genome, especially when genotyping a large number of population samples in breeding work, The cost of a single detection site is very low. It is a highly integrated, high-throughput, miniaturized and automated means to detect SNPs. SNP chip is widely considered as a significant research value by academia and commercial research institutions based on its characteristics of self height flux, automation and high sensitivity, and also has a good trend of industrialization. Well known commercial companies such as ThermoFisher, Agilent, Illumina, Affymetrix (which has been acquired by ThermoFisher and is still named Affymetrix here for better differentiation), among which Illumina's Infinium technology and Affymetrix's Axiom technology should be the most popular high-density SNP chip solutions, so here we will focus on these two technologies.

Illumina Infinium BeadChip

Illumina's biochip system is mainly composed of chips, scanners and analysis software. The chip is composed of glass substrate and bead.

Illumina Infinium chip is a BeadArray biochip based on micro beads. First, on the glass substrate (called Wafer), many aligned holes are etched by means of photolithography, each with a diameter of about 3 μ m. These holes can just hold a bead. Microspheres are the core part of the chip. The surface of each bead is coupled with hundreds of thousands of DNA fragments of the same sequence. These DNA fragments are 73 bases in length and divided into two functional regions. The 23 base sequence near the bead side is called the Address sequence, which is the tag sequence identifying the bead, equivalent to the ID number (ID number) of the bead; The 50 bases far from the bead end are called probe sequences, which are designed for a specific SNP site and are used for complementary hybridization with target DNA. The relationship between Address sequence and probe sequence is one-to-one.

In the production process of Illumina chip, hundreds of thousands of micro beads are mixed according to the set proportion and scattered on the glass substrate. The micro beads randomly fall into the holes of the substrate. Different genotypes are distinguished by detecting the fluorescent signal on the dNTP mixed with the single base extension of DNA fragments on the micro beads.

 

Picture description:

A: Photoetching the glass substrate with a diameter of about 3 μ M, each hole just can hold one micro bead;

B: Several hundred thousand DNA fragments of the same sequence are coupled on the surface of the bead. The 23nt sequence near the bead is the address sequence used to identify the bead, while the 50nt sequence far from the bead end is the probe sequence specific to the SNP site to be tested;

C: Mark C base with green fluorescence and T base with red fluorescence. Take SNP with genotype CT as an example. If the light emitted is green, it is CC homozygous. If the light emitted is red, it is TT homozygous. If both green and red fluorescence exist and the light intensity is similar, then this site is CT heterozygous.
 

 

Illumina's SNP chip has the following advantages:

The detection flux is large, which can detect hundreds of thousands to millions of SNPs at a time;

The detection accuracy is high, which can reach 99.9% or more;

The cost of detection is relatively low. The cost of detection for a 900000 bit chip (each sample) is about RMB 12000. In crop breeding, Illumina Infinium BeadChip technology has designed and developed a large number of SNP chips, such as rice 6K, corn 50K and 3K, wheat 9K and 90K, barley 9K, etc.

Affymetrix AxiomAffymetrix mainly has four models: GeneAtlas, 7G, DX2 and GeneTitan. The two most widely used biochips are expression profile chip and SNP typing chip. SNP chip is also made through "photo etching", that is, DNA probe is synthesized on a large glass substrate, and the 3 'end of the probe is fixed on the glass substrate. Probes of the same sequence are designed for the same SNP.

Affymetrix Axiom genotyping chip is based on linkage reaction for genotyping: firstly, four sets of color probes are designed, one for each group of A, C, G and T, and the length of these probes is 9 bases, of which only the first base at the end of 3 'is specific, and from the second base to the ninth base is degenerate; The 5 'end of a probe with a C or G base at the 3' end bears a biotin label, which will finally be stained with red fluorescence; The 5 'end of probes with A or T base at the 3' end bears another label, which will finally be stained with green fluorescence.

Take an SNP site with genotype GT as an example, first design a specific probe for this SNP site (connected to the chip by hydroxyl group, also known as "capture probe"), which extends to a base next to the SNP site; When the sample DNA is denatured, the single strand DNA will be annealed and hybridized with the capture probe; Add four color probes and connect them through ligase. Because ligase accurately identifies several bases before and after the connection site, only color probes that are completely complementary to the target DNA segment can be connected to the capture probe by ligase; After the connection reaction is completed, the free color developing probe is washed away, and then stained with fluorescent staining reagent; Finally, scan the chip in the laser scanner. If the light emitted from the probe is pure red during scanning, it can be determined that the SNP type of this site is GG homozygous; If the fluorescence emitted is pure green light, it can be determined that this SNP is a TT homozygote; If the light emitted includes both red light and green light, and the light intensity of red light is similar to that of green light, it can be determined that this SNP site is a heterozygote of GT.

 

 

Picture description:

A: DNA probe was directly synthesized on a large glass substrate, and the 3 'end of the probe was fixed on the glass substrate;

B: A color probe with four bases A, T, C and G was designed. The first base at the 3 'end of the probe was specific, and the second to ninth bases were degenerate. C or G would be dyed red fluorescence, while A or T would be dyed green fluorescence;

C: Taking a heterozygote with genotype GT as an example, the probe on the chip extends just to a base in front of the SNP site. Only when it is completely complementary to the target DNA segment, the color probe of base C or A will be connected to the capture probe by ligase.

 

 

Affymetrix Axiom has also designed and developed many representative chips in the field of crop breeding, such as wheat 660K, rice 50K, corn 50K and 600K, and soybean 180K. In addition to Illumina and Affymetrix, Agilent's biochips are also commonly used. The biochip system is similar, that is, it consists of scanner, biochip and analysis software. If you are interested, you can go to the official website to learn about it. Although commercial SNP chips have been widely used in breeding, they still have the following shortcomings:

The number of markers is limited, which is difficult to meet all types of research needs, such as fine positioning of QTL;

Only known mutations can be detected, but new mutations cannot be detected;

Generally, it is designed for some well-known varieties and has a large genetic distance from most of the studied local varieties or closed populations, resulting in the failure of some marker loci in specific populations (for example, the early SNP chip developed based on the sequencing sketch of two temperate maize inbred lines, B73 and Mo17, cannot identify the excellent allelic variation in the tropical maize genome). It is necessary to customize chips for specific varieties and groups, which increases the cost. SNP chips are briefly introduced here. In the next section, we will continue to understand the genome wide SNP typing strategy - genome re sequencing, including simplifying genome sequencing. They make up for the lack of high-density SNP chips to some extent. Coming soon.


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