Method for detecting chromosome deficiencies for congenital abnormality
A technology of chromosome deletion and detection method, which is applied in the field of detection of chromosome deletion in congenital anomalies, and can solve problems such as unexplained congenital anomalies syndrome
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Embodiment 1
[0077] (a) Analysis results using genome disordered arrays
[0078] Genomic disordered arrays are substrates for genomic DNA (BAC clones) that hold regions of human genomic DNA that are deleted or amplified in congenital anomalies.
[0079] Williams Syndrome, Smith-Magenis Syndrome, Down Syndrome, Langer-Giedion Syndrome, Wolf-Hirschhorn Syndrome, Miller-Dieker Syndrome, Prader Willi and Angelman Syndrome can be detected when analyzed using Genomic Disorder Array Syndrome, WAGR Syndrome, Cri du Chat Syndrome, Pallister-Killian Syndrome, Rubinstein-Taybi Syndrome, Hair-Nose-Digital (Toe) Syndrome, Potoki-Shaffer Syndrome, Neurocutaneous I Syndrome, Sotos Syndrome , craniosynostosis syndrome, Kallmann type2 syndrome, Kallmann type1 syndrome, Van der Woude syndrome, ZFHX1B deletion syndrome, Blepharoph imosis and epicanthus syndrome, 1p36 syndrome, cat eye syndrome, Alagille syndrome, Diamond -Blackfan syndrome, congenital adrenal hypoplasia syndrome, steroid sulfatase syndrome,...
Embodiment 2
[0090] [Example 2] Utilize the analysis of Agilent 244K microarray (oligoarray) (Agilent Corporation, USA)
[0091] DNA (0.2 µg each) prepared from patients of Case 1 and Case 2 was analyzed by the CGH method based on Agilent's oligo aCGH microarray 244k type. The 244k type array covers more than 236,000 coding regions and non-coding regions of the human genome, enabling exhaustive analysis of the human genome. The average resolution of the probes was 6.4Kb. The outline of the analysis was carried out according to Agilent's protocol as described below.
[0092] 2.0 μg of DNA from patients and healthy individuals in Case 1 and Case 2 were digested with two restriction enzymes (AluI and RsaI) respectively, and the nick translation method using Exo-Klenow fragments was used. DNA was labeled with Cy5 fluorochrome and DNA from patients was labeled with Cy3 fluorochrome. The labeled DNA was purified using a Microcon YM-30 filter unit (Millipore).
[0093] Heat 158 μl of Cy 3 a...
Embodiment 3
[0095] [Example 3] Analysis based on FISH method
[0096] A hemizygous deletion in the 10q24.31-10q25.1 region was confirmed using FISH in a patient with multiple dysmorphic syndrome with mental retardation. First, lymphocytes were prepared from the blood of two patients, and metaphase chromosomes were produced. That is, human lymphocytes were cultured with 12.5 μg / ml phytohemagglutinin (Phytohemagglutinin) for 3 days, and 0.025 μg / ml colcecamide was added to culture for several hours, the supernatant was removed, 0.075M KCl hypotonic solution was added, and the 30 minutes. Next, the supernatant was removed, and then fixed with Carnot's solution, and the chromosomes were spread on glass slides to prepare metaphase chromosomes.
[0097]BAC DNA (RP11-416N2) containing a hemizygous deletion region (10q24.33) was reacted overnight at 16° C. to take up digoxin-11-dUTP using a nick translation kit. Similarly, as a control, BAC DNA (RP11-357A18) containing the 10q21.2 region not d...
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