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Chen et al. Screening of genetic loci

genome-wide scanning technique. GeneScan -500 LIZ Size Standard, then mixed with
9.5 uL polymerase chain reaction (PCR) product,
METHODS which was diluted 20 times. Tubes containing the
above solution were placed in the PCR instrument
Genomic DNA extraction for degeneration at 95 ℃ for 5 min, kept on ice for
Firstly, 50-100 frozen tissue samples were weighed, more than 5 min. Using an ABI PRISMTM 310 genetic
grinded into powder in liquid nitrogen using a grinding analyzer of the ABI Company for electrophoresis, the
bowel and pestle, and then immediately mixed with voltage was set to 15 KV and run at 60 ℃ for 28 min.
1 mL Tripure. Tissue sample powder was then further The samples were then collected for further analysis.
homogenized 10 times using a homogenizing drill
on ice for 20 min until no tissue fluid particles was Electrophoresis data processing
visible, centrifuged at the speed of 12,000 g at 4 ℃, By using the software Genescan (311) and Genetyper
for 10 min. The homogenate was then kept at room (3.7), we could get the detected fragment size. The
temperature for 5 min to make sure the nuclear protein equipment was provided by ABI Company; the PCR
was totally separated. Each milliliter homogenate was amplification reagents by Baosheng Bioengineering
mixed with 0.3 mL chloroform and shaken vigorously Limited Company; and the fluorescent primers by
at 4 ℃ for 15 s, kept at room temperature for 2-15 min, Shanghai Jikang Biotechnology Limited Company.
then Centrifuged at 12,000 g at 4 ℃, for 15 min. To get
high quality DNA, the upper layer of colorless aqueous RESULTS
liquid after centrifugation was removed. Each milliliter
homogenate was mixed with 0.2 mL 100% ethanol Scanning 12 microsatellite spots on the 17th
which was stored at 4 ℃, mixed completely by rotating chromosome of two mouse lines
the bottle upside down several times and kept at room The differences of 12 microsatellite loci between two
temperature for 2-3 min for DNA precipitation. After inbred mouse lines BALB/C and C57BL/6 were first
centrifuging at 2,000 g for 5 min at 4 ℃, the upper scanned. Among the loci scanned, seven of them
layer liquid was removed with a pipette carefully. Each were significantly different: D17MIT245.1, D17MIT46,
milliliter sample solution was then mixed with 1 mL of D17MIT51.1, D17MIT180.1, D17MIT20. 1, D17MIT184
0.1 mol/L sodium citrate dissolved in 10% ethanol, kept and D17MIT39.1 [Table 3; Figure 1].
at room temperature for 30 min with frequent mixing,
and centrifuged at 4 ℃ for 5 min at 2,000 g again. Scanning microsatellite loci on the 17th
The upper layer liquid was collected, mixed with 75% chromosome using three inbred mouse lines
ethanol, and kept at room temperature for 30 min with To minimize false-positives among the above seven sites
frequent mixing. Then, after centrifuging at 4 ℃ and obtained using the two inbred mouse lines, we further
2,000 g for 5 min, the upper layer liquid was removed searched the literature and found that another inbred
and the DNA sample was dried in the air or vacuum for mouse line, DBA-2, had similar susceptibility to HSV
5-10 min. Finally, we dissolved the DNA with 50 uL TE infection as BALB/C mice. Therefore, scanning these
solution, pipetted out 1 uL sample for color comparison three inbred mouse lines for microsatellite loci led to the
and another 10 uL for electrophoresis, and the residual exclusion of two of the above seven loci, D17MIT245.1
was stored at -20 ℃ for further analysis. DNA samples and D17MIT46. Our updated scanning results showed
were diluted with 90 uL MQ water and analyzed with that D17MIT51.1, D17MIT39.1 and the genomic region
ultraviolet spectrophotometer. The OD260 value, between D17MIT180.1 and D17MIT184 were mouse
OD280 value and OD260/280 value were used for microsatellite regions affecting susceptibility to HSV
calculating the concentration of DNA. infection [Table 4; Figure 2].

Primer design and synthesis Bioinformatic analysis of genes in the
Primer design referenced information from the mouse HSV infection susceptibility regions on
genome program (detailed information can be viewed chromosome 17
on the website of Jackson Laboratory), which was For identifying potential genes involved in the
designed by Shanghai Jikang Biotechnology Limited susceptibility of mice to HSV infection, we used
Company [Table 1]. The detailed information about bioinformatics to analyze the genes localized in these
PCR reaction system and PCR reaction condition can regions. Based on the above results, bioinformatic
be seen in Table 2. analysis found approximately 140 genes in the
positive sites D17MIT51.1, D17MIT39.1 and the region
Microsatellite loci detection between D17MIT180.1 and D17MIT184 [Tables 5-7].
First, 1 mL Hi-Di Formamide was mixed with 50 uL Among those genes, there were about 33 human
Neuroimmunology and Neuroinflammation ¦ Volume 3 ¦ December 26, 2016 275