Gene Product Sequence Variation Clinical Trial
Official title:
Candidate Gene Screening for 6-14 Year Old Patients With ADHD (Attention Deficit/ Hyperactivity Disorder)
Source: Sample bank of Xijing Hospital and Children's Hospital Affiliated to Soochow
University; Sample form: Whole blood; Estimated number of samples: 100 patients with ADHD and
age, sex matched healthy controls; Case exclusion criteria: all kinds of neuropsychiatric
disorders, IQ value of less than 70;
Study protocol:
1. Using Qiagen kit to extract the genomic DNA of 200 microliters of blood.
2. UV spectrophotometer test DNA purity of 260/280 close to 1.8 (1.8 ± 0.05), the
concentration of 100ng/μL or more before the next sequencing.
3. The extracted genomic DNA will be sent to Sangon Biology Engineering Limited Company
(Shanghai) and sequenced to find candidate mutations related to ADHD risk sequence.
According to NIH gene database, the longest transcript of NDRG2 (ID: 57447 gene,
https://www.ncbi.nlm.nih.gov/nuccore/NC_000014.9?
Report=genbank&from=21016763&to=21070872&strand=true) (a total of 17 exons and 16
introns and the gene 5 'UTR and 3' UTR region) will be alignmented sequences to find
potential mutations.
4. Using the chi square analysis and other statistical methods to determine the
relationship between the mutations and susceptibility to ADHD.
Source: Sample bank of Xijing Hospital and Children's Hospital Affiliated to Soochow
University; Sample form: Whole blood; Estimated number of samples: 100 patients with ADHD and
age, sex matched healthy controls; Case exclusion criteria: all kinds of neuropsychiatric
disorders, IQ value of less than 70;
Study protocol:
1. Using Qiagen kit to extract the genomic DNA of 200 microliters of blood. (1) melt frozen
blood at room temperature; (2) take the blood of 0.2 ml into a sterile anti coagulation
centrifuge tube, add an equal volume of PBS phosphate buffer, after fully mixing
12000rpm centrifugation 5min, discard supernatant; (3) 66.7 L STE 2.4 L, 20% SDS, 37 DEG
C water bath 1h; (4) protease K plus 1 20mg/ml l mix, 55 DEG C water bath digestion
overnight (10 ~ 14h); (5) the digested samples treated with Tris saturated phenol, shake
well, 12000rpm centrifugal 10min; (6) the upper aqueous phase to a sterile centrifuge
tube; adding volume of Tris saturated phenol, shake well, 12000rpm centrifugal 10min;
(7) the upper aqueous phase was transferred to another sterile centrifuge tube, and an
equal volume of phenol was added: chloroform: isoamyl alcohol (25: 24: 1). The
oscillation of the vortex was fully mixed, and the 12000rpm was centrifuged to 10min;
(8) the upper aqueous phase was transferred to another sterile centrifuge tube, and an
equal volume of chloroform was added: iso amyl alcohol (24: 1), which was fully vibrated
and mixed with 12000rpm centrifugation 10min; (9) transfer the supernatant to another
sterile centrifuge tube; adding 2 times volume ethanol, the volume level of sodium
acetate 1/10 shake, until the flocculent precipitation of DNA visible; (10) the sample
is placed at -20 DEG C refrigerator freezer 30min or ice bath for 15 ~ 20min, after
removal of 12000rpm centrifugal 10min again, so that DNA precipitation; (11) the gun
head will pick DNA to another sterile centrifugal tube, with the amount of 70% ethanol
washing and shaking, to wash out impurities DNA; (12) out of ethanol, DNA by vacuum
drying or dry naturally, TE buffer adding dissolution, -20 stored at standby.
2. UV spectrophotometer test DNA purity of 260/280 close to 1.8 (1.8 ± 0.05), the
concentration of 100ng/μL or more before the next sequencing.
3. The extracted genomic DNA will be sent to Sangon Biology Engineering Limited Company
(Shanghai) and sequenced to find candidate mutations related to ADHD risk sequence.
According to NIH gene database, the longest transcript of NDRG2 (ID: 57447 gene,
https://www.ncbi.nlm.nih.gov/nuccore/NC_000014.9?
Report=genbank&from=21016763&to=21070872&strand=true) (a total of 17 exons and 16
introns and the gene 5 'UTR and 3' UTR region) will be alignmented sequences to find
potential mutations.
4. Using the chi square analysis and other statistical methods to determine the
relationship between the mutations and susceptibility to ADHD.
;
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