Abstract
This study aimed to establish a method for the selective amplification of cell-free fetal DNA (cffDNA) in maternal plasma and preserve the integrity of DNA fragments during amplification, thereby providing a sufficient amount of cffDNA to meet the requirement of routine non-invasive prenatal testing. We amplified DNA molecules in a one-reaction system without considering their particular sequences and lengths (overall amplification) by using PCR-based enrichment. We then modified PCR conditions to verify the effect of denaturation temperature on DNA amplification on various lengths of DNA (selective overall amplification). Finally, we used an optimum temperature range to amplify cffDNA selectively. Amplification results were validated by electrophoresis and real-time quantitative PCR. Our PCR-based enrichment efficiently amplified all DNA fragments with differing lengths within a single reaction system, as well as preserving the integrity of the DNA fragments. cffDNA was significantly amplified along with the selective amplification of small fragment maternal plasma DNA in an appropriate range of denaturation temperatures. We have established a PCR-based method for the simultaneous enrichment and amplification of cffDNA in order to meet the requirements of high cffDNA quantity for routine non-invasive prenatal testing.
Highlights
The discovery of cell-free fetal DNA in maternal plasma has greatly promoted the development of non-invasive prenatal diagnosis[1]
All cell-free fetal DNA (cffDNA) fragments are shorter than 300 bp and approximately 20% of maternal cfDNA fragments are longer than 300 bp[5,6,7,8]
When the denaturation temperature was increased to the range of 82.4–83.0 °C, both T-2 and T-3 group were amplified and showed the expected bands (Fig. 5A and B). These results suggest that reducing the denaturation temperature of the PCR within a certain range can impede the amplification of large DNA fragments without affecting the amplification of small DNA fragments, and the temperature which impedes the amplification of DNA fragments larger than 300 bp may be within the range of 79.9–82.4 °C
Summary
The discovery of cell-free fetal DNA (cffDNA) in maternal plasma has greatly promoted the development of non-invasive prenatal diagnosis[1]. CffDNA is mixed with maternal derived cell-free DNA, and current studies on cffDNA are carried out under the background interference of large amounts of maternal derived cell-free DNA These limitations restrict the application of cffDNA in clinical testing or diagnosis. The purpose of this study was to overcome the problems associated with existing DNA amplification technologies and establish a PCR-based enrichment protocol for the selective amplification of cffDNA by modifying amplification reaction conditions of AFLP. This established method is sufficient to provide a large amount of cffDNA to meet the requirement of routine testing
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
