Microsatellites, also known as simple tandem repeats (STRs), are simple repeat sequences distributing in eukaryotic genomes, which are composed of 2 ~ 6 nucleotide tandem repeats. The repeating times in individual are variable, therefore, the application of microsatellite markers is very extensive. Microsatellite loci is usually amplified by PCR. PCR products are analyzed by electrophoresis and separate alleles according to size for detection. PCR amplified alleles can be detected in a variety of ways, such as fluorescent staining or silver staining.
Microsatellites exist in most biological genomes. They are evenly distributed in the genome, so they are widely used in genetic cross breeding and mapping markers of chromosome genetic map. Highly polymorphic microsatellite markers can be used to identify individuals in the population.
Since the application of microsatellite is so wide, microsatellite research has become especially important. The following is a detailed introduction of some microsatellite genotyping procedures, which are totally different from those of SNP Genotyping we may talk later.
Primers design and synthesis
Primer design is that a small segment of single stranded DNA or RNA, as the origin of DNA replication, plays a role in nucleic acid synthesis reaction, as a polynucleotide chain. In the 3′-OH primer, nucleotide is synthesized in two ester chain form, so the 3′ -OH of primer must be free.
At present, the primers are synthesized by solid phase three ester method. The DNA fragment can be synthesized by that method with characteristics of high efficiency, rapid coupling and stable initial reaction. The method immobilizes DNA on solid phase for synthesis, and the synthesis direction is from 3 ‘end to the 5’ end. The adjacent nucleotides are connected by 3-5 ester phosphate bond.
Characteristics of primer synthesis
1. the purity of the synthesized primer is high;
2. synthetic sequence length is long;
3. the effect of synthesis is good.
Receipt of gDNA
gDNA (genomic DNA): refers to the total content of DNA in haploid state. The generalized genome refers to the DNA in a system (such as nuclear or organelle), which includes coding or inherent ribosomal DNA (rDNA) in cells, mitochondrial DNA (mtDNA), tRNA gene and other RNA coding.
gDNA quality assessment
Quantitative analysis and integrity assessment of genomic DNA is an important part of genetic analysis based on an accurate and reliable chip or high-throughput sequencing technology. It is also a rapid and accurate objective link analysis technology. gDNA assay and GQS—genomic DNA quality score can solve this problem effectively. It makes the LabChip GX biological macromolecular analyzer based on the assay become a technology platform with wide application, largest sensitivity and flux, economic cost, data repeatability and best automation standardization in nucleic acid sample analysis control field.
Pooling of amplified PCR products for multiple loading
The basic principle of PCR technology is similar to the natural replication process of DNA, and its specificity is dependent on the oligonucleotide primers complementary to the two ends of the target sequence.
PCR is an in vitro DNA amplification technology. It is the multiple cycle of “high- temperature annealing, low-temperature denaturation and primer extension” for amplified DNA fragment and two complementary oligonucleotide primers in the conditions of template DNA, primers and 4 kinds of DNA polymerase, increasing the number of DNA fragments exponentially, thus obtaining a large number of specific gene segments we need in a short period of time.
Capillary electrophoresis of pooled PCR products
Simple and fast PCR reaction uses thermostable Taq DNA polymerase. Once the reaction liquid is added, it can carry out the experiment of denaturation-annealing-extension reaction on DNA amplification liquid. Usually 2 to 4 hours are needed to complete the reaction. The amplified products are analyzed by electrophoresis instead of isotope to avoid radioactive contamination.
The purity of the sample is not required. The virus or bacteria and culture cells are not needed to be isolated. DNA crude products and total RNA can be used as amplification templates. They can be used directly in clinical samples, such as the detection of blood, body fluid, wash fluid, hair, living tissue cells and crude DNA amplification.
In environmental detection, target nucleic acid sequences often exist in a complex mixture such as cell extracts, and the content is very low. For the specific detection of microbes and genes in this complex microbial group, hybridization is not sensitive. PCR technology can be used to amplify the target sequence with several orders of magnitude, and then probe hybridization detects the amplified sequence for qualitative or quantitative research and analysis of microbial population structure. PCR technology and other technologies are used in combination, such as RT-PCR, RAPf, nested PCR and ARDRA.
Genotyping analysis using AB GeneMapper software
GeneMapper use-method: getting starded-setting up the microsatellite analysis-analyzing and examining results-printing and exporting results.
Compilation of results
Finally, the original data of the primer, the microsatellite analysis, the PDF map, the GeneMapper map, the length information, the Excel and the results of the analysis are provided.
Next blog it will introduces something about the metagenomics sequencing.