OBJECTIVE: Among the pitfalls of using cell-free fetal DNA in plasma for prenatal diagnosis is quality of the recovered DNA fragments and concomitant presence of maternal DNA (>95%). Our objective is to provide alternative methods for achieving enrichment and high-quality fetal DNA from plasma. METHODS: Cell-free DNA from 31 pregnant women and 18 controls (10 males and 8 females) were size separated using agarose gel electrophoresis. DNA fragments of 100-300, 500-700 and 1,500-2,000 bp were excised and extracted, followed by whole genome amplification (WGA) of recovered fragments. Levels of beta-globin and DYS1 were measured. RESULTS: Distribution of beta-globin size fragments was similar among pregnant women and controls. Among control male cases, distribution of size fragments was the same for both beta-globin and DYS1. Among maternal cases confirmed to be male, the smallest size fragment (100-300 bp) accounted for nearly 50% (39.76 +/- 17.55%) of the recovered DYS1-DNA (fetal) and only 10% (10.40 +/- 6.49%) of beta-globin (total) DNA. After WGA of plasma fragments from pregnant women, DYS1 sequence amplification was best observed when using the 100-300 bp fragments as template. CONCLUSIONS: Combination of electrophoresis for size separation and WGA led to enriched fetal DNA from plasma. This novel combination of strategies is more likely to permit universal clinical applications of cell-free fetal DNA. Copyright 2009 S. Karger AG, Basel.
OBJECTIVE: Among the pitfalls of using cell-free fetal DNA in plasma for prenatal diagnosis is quality of the recovered DNA fragments and concomitant presence of maternal DNA (>95%). Our objective is to provide alternative methods for achieving enrichment and high-quality fetal DNA from plasma. METHODS: Cell-free DNA from 31 pregnant women and 18 controls (10 males and 8 females) were size separated using agarose gel electrophoresis. DNA fragments of 100-300, 500-700 and 1,500-2,000 bp were excised and extracted, followed by whole genome amplification (WGA) of recovered fragments. Levels of beta-globin and DYS1 were measured. RESULTS: Distribution of beta-globin size fragments was similar among pregnant women and controls. Among control male cases, distribution of size fragments was the same for both beta-globin and DYS1. Among maternal cases confirmed to be male, the smallest size fragment (100-300 bp) accounted for nearly 50% (39.76 +/- 17.55%) of the recovered DYS1-DNA (fetal) and only 10% (10.40 +/- 6.49%) of beta-globin (total) DNA. After WGA of plasma fragments from pregnant women, DYS1 sequence amplification was best observed when using the 100-300 bp fragments as template. CONCLUSIONS: Combination of electrophoresis for size separation and WGA led to enriched fetal DNA from plasma. This novel combination of strategies is more likely to permit universal clinical applications of cell-free fetal DNA. Copyright 2009 S. Karger AG, Basel.
Authors: Y M Lo; M S Tein; T K Lau; C J Haines; T N Leung; P M Poon; J S Wainscoat; P J Johnson; A M Chang; N M Hjelm Journal: Am J Hum Genet Date: 1998-04 Impact factor: 11.025
Authors: Aaron F Orozco; Farideh Z Bischoff; Cassandra Horne; Edwina Popek; Joe Leigh Simpson; Dorothy E Lewis Journal: Ann N Y Acad Sci Date: 2006-09 Impact factor: 5.691
Authors: Carolina J Jorgez; Dianne D Dang; Joe Leigh Simpson; Dorothy E Lewis; Farideh Z Bischoff Journal: Genet Med Date: 2006-10 Impact factor: 8.822
Authors: José Luis García-Giménez; Marta Seco-Cervera; Trygve O Tollefsbol; Carlos Romá-Mateo; Lorena Peiró-Chova; Pablo Lapunzina; Federico V Pallardó Journal: Crit Rev Clin Lab Sci Date: 2017-12-11 Impact factor: 6.250
Authors: Alison S Devonshire; Alexandra S Whale; Alice Gutteridge; Gerwyn Jones; Simon Cowen; Carole A Foy; Jim F Huggett Journal: Anal Bioanal Chem Date: 2014-05-24 Impact factor: 4.142
Authors: Sabine Hellwig; David A Nix; Keith M Gligorich; John M O'Shea; Alun Thomas; Carrie L Fuertes; Preetida J Bhetariya; Gabor T Marth; Mary P Bronner; Hunter R Underhill Journal: PLoS One Date: 2018-07-25 Impact factor: 3.240