Evaluating the Efficiency of REPLI-g® Single Cell Kit for Trace DNA Amplification / 法医学杂志

Objective To evaluate the efficiency of REPLI-g® Single Cell Kit for sample DNA amplification, and explore its application value in forensic trace DNA amplification. Methods Three DNA extraction kits were selected to extract DNA from peripheral blood of 10 unrelated individuals. The DNA yield and purity of the three DNA extraction kits were compared. According to the results of comparison, one DNA sample was selected to concentrate and dilute, then used as the initial sample of whole genome amplification （WGA）. REPLI-g® Single Cell Kit was used to amplify the initial sample at the whole genome level. The amplification yield and amplification times were calculated, and the distribution of DNA fragments was detected by agarose gel electrophoresis. Goldeneye® DNA ID System 20A Kit was used to perform the STR typing of the initial sample and DNA samples amplified at the whole genome level to evaluate the performance of REPLI-g® Single Cell Kit in trace DNA amplication in terms of purity and yield as well as the success rate of STR typing. Results After comparison, one DNA sample was selected from QIAsymphony® DNA Investigator® Kit extracts to concentrate and dilute as the initial sample of WGA. After amplifying the whole genome of a series of initial samples by REPLI-g® Single Cell Kit, the lowest average of amplification yield reached 8.77×103 ng, while the average of the corresponding amplification times reached 1.40×106. DNA fragments were large and concentrated. The STR typing success rate of WGA samples became lower with the decrease of initial samples used, but when the amount of samples was lower than 0.5 ng, the STR typing success rate of samples after DNA WGA was higher than that of samples without DNA WGA. Conclusion REPLI-g® Single Cell Kit can increase the yield of template DNA. Especially for trace DNA, the STR typing success rate can be improved to a certain extent.

Single cell-based molecular detecting technologies in preimplantation genetic testing / 中华检验医学杂志

Since the first report of preimplantation genetic diagnosis success in clinical in 1989,it is technological development that has been the driving force of preimplantation genetic testing(PGT).As the early stage prenatal diagnosis, PGT is used in the setting of assisted reproduction to differentiate the genetically normal embryos from pathogenic mutations or chromosomal anomalies in order to increase the rates of fruitful pregnancy.PGT involves technologies of assisted reproduction, molecular detection and genetic counseling.The aim of this review is to, from the perspective of laboratory medicine, summarize the utilization and progress of molecular strategies in the field of PGT,and explore its future development.(Chin J Lab Med,2018,41:270-274)

The application of whole genome amplification in DNA typing of trace samples / 中国法医学杂志

Objective To explore the effectiveness of whole genome amplification technology in DNA typing of trace samples.Methods Simulated trace samples which contain 1～20 cells were prepared by micromanipulation.Whole genome amplification was added before conventional PCR-STR typing step,to compare the effectiveness of PEP and MDA in DNA typing of trace samples from four aspects i.e.allele imbalance,allele drop-out,locus drop-out and pseudo allele (which contains the stutter peak).Results Amplification efficiency of MDA was higher than PEP method,but allele drop-out and pseudo allele were more frequently detected.Correct DNA typing rate of PEP is higher than MDA method,however,advantaged amplification of small fiagments DNA is more obvious.Conclusion MDA method is not suitable for the current STR typing.When the absolute amount of trace samples is quite small,we couldconsider using the PEP method to enhancethe sample quantity to meet the requirement of repeat testing.At the same time it could encounterthe failure of the large DNA fragments.

Prenatal diagnosis of chromosomal abnormalities using whole genome amplification / 实用医学杂志

Objective To evaluate the value of whole genome amplification (WGA) combined with array comparative genomic hybridization (aCGH) in prenatal diagnosis. Methods Array CGH were performed by the DNA of 18 prenatal specimens , which were amplified by WGA because of the low DNA yield. Result 3 of the 18 fetuses were 45, X0 and 9 of 15 fetuses with normal aCGH results showed healthy outcome. Conclusion It’ s feasible for prenatal diagnosis using WGA combined with aCGH which not only can shorten the reporting time but also keep the sensitivity and accuracy of detection.

An update of preimplantation genetic diagnosis in gene diseases, chromosomal translocation, and aneuploidy screening / 대한생식의학회지

Preimplantation genetic diagnosis (PGD) is gradually widely used in prevention of gene diseases and chromosomal abnormalities. Much improvement has been achieved in biopsy technique and molecular diagnosis. Blastocyst biopsy can increase diagnostic accuracy and reduce allele dropout. It is cost-effective and currently plays an important role. Whole genome amplification permits subsequent individual detection of multiple gene loci and screening all 23 pairs of chromosomes. For PGD of chromosomal translocation, fluorescence in-situ hybridization (FISH) is traditionally used, but with technical difficulty. Array comparative genomic hybridization (CGH) can detect translocation and 23 pairs of chromosomes that may replace FISH. Single nucleotide polymorphisms array with haplotyping can further distinguish between normal chromosomes and balanced translocation. PGD may shorten time to conceive and reduce miscarriage for patients with chromosomal translocation. PGD has a potential value for mitochondrial diseases. Preimplantation genetic haplotyping has been applied for unknown mutation sites of single gene disease. Preimplantation genetic screening (PGS) using limited FISH probes in the cleavage-stage embryo did not increase live birth rates for patients with advanced maternal age, unexplained recurrent abortions, and repeated implantation failure. Polar body and blastocyst biopsy may circumvent the problem of mosaicism. PGS using blastocyst biopsy and array CGH is encouraging and merit further studies. Cryopreservation of biopsied blastocysts instead of fresh transfer permits sufficient time for transportation and genetic analysis. Cryopreservation of embryos may avoid ovarian hyperstimulation syndrome and possible suboptimal endometrium.