The Establishment Of EL-MLPA Technology

Objective:①Studying and establishing a method can detect trisomy 21 syndrome (D.S)critical region of short tandem repeat (STR) allele copy number on 21 chromosome.②Discovery trisomy 21 syndrome feasibility of noninvasive prenatal screening.Methods:①Based on the MLPA technology, we select 8 high heterozygosity of STRsequence which has high heterozygosity on 21 chromosome in Chinese people and synthetic 8probes which need to be used by EL - MLPA.②In the same reaction tube, left probe isextensive and connect with right probe simultaneously. That is selecting the most suitable DNApolymerase (AmpliTaq DNA Polymerase, stoffel Fragment) and ligase (Ampligase, SALSALigase-65) and jointing them for extension and ligation at the same time to establish theEL-MLPA technology.③Extraction of human DNA in peripheral blood: this experiment useQIAamp DNA Blood Mini Kits of Qiagene company to extract DNA, use DHPLC (denaturinghigh performance liquid chromatography) instrument to detect the purity and use cytogenetickaryotype analysis to confirm the nuclear type.④We use the EL-MLPA technique toquantitative detect 8 STR polymorphisms allele on 21 chromosome.⑤The results wereseparated and analyzed by ABI 3130XL, and compared with cytology karyotype analying resultsso as to verify the feasibility and stability of this technology.Result: With the set up of EL-MLPA, 80 copies of human peripheral blood DNA aredetected, including 50 normal DNA and 30 patients with 21– trisome, separated and analyzed byABI 3130XL. The results show that detecting DNA from normal patterns appears a toweringpeak or two peaks, with peak height close. Detection of 21 - trisomy DNA appears two peaks,one peak height is about two times to another or three peaks, with peak height close. Test resultsand karyotype analysis results are consistent.Conclusion:①EL-MLPA technology is a good method to detect STR alleles copy numberon chromosome 21.②It can accurately measure the target gene STR alleles copy number0.5-1.0 times increase or decrease, and can be used for trisomy 21 fetal's prenatal screening. Objective: To found a simple and rapid method for detecting common chromosomeaneuploidies by using multiplex ligation-dependent probe amplification(MLPA) / denaturinghigh pressure liquid chromatograghy(DHPLC) techniques.Methods:①By using QIAamp DNA Blood Mini kits to extract human DNA in peripheralblood, by DHPLC (denaturing high performance liquid chromatography) instrument to detect thepurity and using cytogenetic karyotype analysis to confirm the karyotype.②Using SALSAMLPA Kit-P095, DNA samples will be tested MLPA reaction, Hybrid - Connect-PCR-Gelelectrophoresis.③The MLPA reaction products of DNA sample were separated and analyzedby DHPLC, and the results obtained were compared with that determined by ABI 3130XL.Result: DNA samples from 10 normal people,16 patients with 21-trisome,3 patients with47,XYY,3 patients with 47,XXX were tested by MLPA. PCR reaction products were detectedby DHPLC instrument. The results show that compared with normal DNA, 21 - trisomy DNAincomes, representatives from atlas of the 21st chromosome were significantly increased,corresponding to normal DNA all peak figure increase high about 50%. 47, XYY DNA profiles,the four peaks that representing Y chromosome increased significantly, the peak heigh is abouttwo times of the normal human DNA. 47, XXX DNA map, eight peaks on behalf of Xchromosome are significantly increased. The results obtained are identical with the resultsobtained by ABI 3130XL and the results of Karyotyping.Conclusion:MLPA reaction products with single pipe combines DHPLC could be used torapidly and accuratly identify the common chromosome aneuploidies.