Establish A Method Of Cell-free Fetal DNA In Maternal Plasma Selective Amplification And Its Application In Trisomy21Noninvasive Prenatal Testing

In China, the annual birth defects up to800,000to1,200,000, birth defectsoccur in approximately4%to6%, which makes a heavy mental stress and financialburden to the family and society. For now, the most effective countermeasures againstbirth defects is check screening and diagnosis as early as possible, timely terminationof pregnancy process after detection and diagnosis is an effective way to reduce theincidence of birth defects. Current conventional methods are invasive prenataldiagnosis such as amniotic fluid cytology, chorionic villus sampling and fetal cordblood test which have a certain degree of risk to pregnant women and fetuses. And thesensitivity and accuracy of noninvasive prenatal detection method such as serologicalscreening and ultrasound are poor. Therefore, the development of new noninvasiveprenatal testing method has important clinical implications, and has been one of thefocuses of the international medical genetics and reproductive medical profession.Since the present of cell-free fetal DNA (cffDNA) in maternal plasma andserum was confirmed by Lo et al in1997, it provides a new way for non-invasiveprenatal testing (NIPT). In healthy gravidae, cffDNA can be detected in maternalplasma as early as the7th week after conception, then increases by about21%perweek with the pregnancy progresses, reaches the plateau in the ensuing three months,rapid increase again before delivery, ultimately promptly cleared from maternalplasma and disappears within two hours of delivery. CffDNA can be used for earlyand specifically prenatal screening and diagnosis without disturbed by the previouspregnancy. These features make cffDNA become the ideal samples for NIPT. But due to the cffDNA contained in maternal plasma is extremely low, only approximately19%of maternal plasma total cell-free DNA (cfDNA), it is easy to lose in the extractionprocess, increased the difficulty of extraction and decreased the sensitivity of testing.Furthermore, because the cffDNA is mixed with the maternal derived cell-free DNA,current studies on cffDNA are carried out under the background interference ofmassive maternal derived cell-free DNA and it is difficult to avoid the inaccurateresults. And in recent years, the next generation sequencing technology is receivingmuch concern, but its high price limits its universal application in clinical detection.It will be very meaningful for the clinical application of cffDNA if we caneliminate the background interference of maternal derived cell-free DNA, increase thecontent of cffDNA. Only a breakthrough in cffDNA enrichment method, in order toeffectively improve the accuracy of diagnosis, therefore, a new method for enrichingand obtaining abundant cffDNA that can meet the demands of routine testing isrequired.Chan et al found that, the vast majority of studies support that cffDNAmolecules are generally shorter than300bps while the maternal-derived ones arelonger than300bps. According to this characteristic, it is possible to enrichmentcffDNA by collecting the maternal plasma cell-free DNA fragments whose length isshorter than300bps. Current methods for enrichment cffDNA is separating the shortfragments cffDNA from large fragments maternal derived cell-free DNA throughelectrophoresis, but the precision of this method is extremely low and it is easy tointroduce exogenous nucleic acid contaminants. Domestic and international, there isno efficient and specifically method for enrichment and separation cffDNA frommaternal plasma, thereby performing noninvasive prenatal testing.Amplified fragment length polymorphism (AFLP), is a PCR-based techniquethat can amplify restriction fragments selectively. The basic procedure is firstly,digestion of genomic DNA with one or more restriction enzymes and then ligation ofrestriction half-site specific linkers to the sticky ends of all restriction fragments,finally, amplification of these fragments with two primers that complementary to thelinker and restriction site specific sequences. Advantage of this principle, cffDNA can be overall amplification through ligating specific linkers to both ends of cffDNAmolecules.The denature temperature (Tm) of DNA molecule is related to a number offactors such as molecule length, base composition, ionic strength of the buffer. If theTm is reduced in an appropriate range, the amplification of larger DNA fragments willbe suppressed without affecting on the amplification of smaller DNA fragments.Applying this principle, smaller DNA fragments can be amplified selectively byreducing the Tm.The subject improved the conventional AFLP technique based on its principle.Establish a method of selectively amplify cffDNA from maternal plasma whileretaining the integrity of DNA fragments by optimizing the overall amplificationreaction conditions and reaction system. This method lays an experimental foundationfor NIPT using cffDNA. Finally, establish a new method for the NIPT of trisomy21syndrome with the selectively overall amplified cffDNA.This study is divided into four parts:1. Comparison of plasma DNA extraction methods. The cfDNA was extractedby once/twice phenol-chloroform-isoamyl alcohol extraction, once/twicephenol-chloroform-isoamyl alcohol extraction combined with DNAdown, andmesoporous nano-beads methods. And then determine the extraction efficacy of eachmethod.2. Establish the method of selective overall amplification of cell-free fetal DNAbased on AFLP. Based on the principle of AFLP technique, ligate the pretreatedcell-free plasma DNA with double stranded unidirectional linkers, then amplified theligation product with a universal primer which is complementary to the linker in orderto overall amplify all the DNA fragments. According to the relationship between Tmand molecule length, base composition, ionic strength and other factors, optimizingthe overall amplification reaction conditions and reaction system, establish a methodof selectively amplify specific length of the template. Finally, using this methodselectively amplify the DNA fragments which is less than300bps from maternalplasma, establish a method of selectively amplify cffDNA from maternal plasma while retaining the integrity of DNA fragments.3. Evaluation and validation of suitable control genes for real-time quantitativePCR studies in pregnant plasma DNA. Examine a panel of six common control genes(HBB, TERT, GAPDH, ALB, ACTB and TRG) in order to evaluate and validate themost reliable control genes for qPCR studies in the quantitation of pregnant femalesplasma DNA, non pregnant females plasma DNA, maternal plasma DNA moleculeslager than300bps (considered as maternal-derived DNA) and smaller than300bps(considered as fetal-derived DNA).4. Establish a method for the NIPT of trisomy21syndrome based on AFLP. ThecffDNA in maternal plasma obtained through selectively amplification was used assubstrates for detection, combined four kinds of chromosome21specific recognitionfragment as target genes, qPCR was used to detect the relative content of chromosome21, establish a linear discriminant function through discriminant analysis todiscriminate predicted normal and trisomy21syndrome. Finally establish a methodfor the NIPT of trisomy21syndrome.The conclusions are as follows:1. Comparison of five methods of plasma DNA extraction, proofed that oncephenol-chloroform-isoamyl alcohol extraction combined with DNAdown method islow cost, extraction yield, less DNA damage, high purity, but the operation is morecomplicated and time consuming; the cost of mesoporous nano-beads method isrelatively slightly higher, but the operation is simple and time consuming is short.Provide the basis for a method of extraction cell-free DNA from plasma.2. Establish a method for selective amplification of small double-stranded DNAmolecules (<300bps) in maternal plasma. Achieve enrichment cffDNA fraction whileamplified greatly; reduce background interference from cell-free DNA in maternalplasma. This method lays an experimental foundation for the use of cffDNA in NIPT.3. The content stability of six common control genes are evaluated and validatedin pregnant females plasma DNA, non pregnant females plasma cell-free DNA anddifferent length of maternal plasma DNA molecules. The content stabilities of controlgenes were proved to be a significant difference in plasma cell-free DNA. In pregnant plasma DNA compare non-pregnant plasma DNA group, pregnant plasma DNA group,non-pregnant plasma DNA group, maternal-derived DNA compare fetal-derived DNAgroup, maternal-derived DNA group and fetal-derived DNA group, the genes withhighest content stability are TERT, ACTB, ALB, HBB, HBB and GAPDHrespectively. These results provide a theoretical basis for the use of maternal plasmacell-free DNA in relative quantification molecular biology experiments.4. Establish a method for earlier, high accuracy, low risk and cost effectivedetection of trisomy21syndrome through the improved AFLP and qPCR technology.This method lays a methodological foundation for the high-throughput synchronousscreening of various chromosome aneuploidy disease and monogenic diseases.In this subject, we applied the AFLP technique; selectively amplify the traceamounts of cffDNA from maternal plasma while retaining the integrity of DNAfragments, reduce background interference from cell-free DNA in maternal plasma,breakthrough the restriction of difficulty in obtaining samples for the application ofcffDNA in NIPT, establish a new method for detection of trisomy21syndrome basedon AFLP technique. This method has earlier detection, high accuracy, low risk, costeffective and other characteristics. Advantage of this technique, a number of otherhigh-throughput synchronous screening NIPT of various chromosome aneuploidydisease and monogenic diseases can be achieved.