2.4. Multiplex quantitative fluorescent polymerase chain reaction (MQF‐PCR)

Genomic DNA from 200 μL peripheral blood of the propositus, her family members, a healthy fertile man, and a normal woman was extracted using blood DNA extraction Kits (TIANGEN, Beijing, China), using the procedure recommended by the producer. Amplification on 2 STR loci on chromosomes X (DXS981, DXS6809), one common STR loci on chromosomes X and Y, one gender specific loci AMXY, and three STR loci on chromosome 21 specific (D21S1435 D21S1411, D21S11) was conducted using 21‐trisome and sex chromosomes polyploid detection kit (Daan Gene, Guangzhou, China), PCR mix created as directed by the producer. The kit contains primer pairs for 4 markers on sex chromosomes, 3 markers for chromosome 21, all in a single multiplex reaction. Fragmental Analysis was conducted on an ABI 3130 Genetic Analyzer, using Run 3130 Data Collection software, using 36 cm capillary array length, and performance optimizing polymer (POP) 7. Run time was set to 1800 seconds. Sizing standard used was ABI LIZ 500. Data analysis and electropherogram creation were carried out using GeneMapper ID v3.2 software.