Study Of Method To Enrich Fatal Dna In Maternal Plasma With Immunology Magnetic Beads

Background: Many kinds of hereditary disease such as hemolytic disease of new born, hemophilia, albinism, thalassemia, and phenyl ketonuria do greate harm to patients, yet we have no good methods to give radical treatment. To find a pragmatic method to give accurate dignosis in early pregnancy has greate clinic significance. Traditional methods for prenatal diagnosis consist centesis to amniotic cavity, sampling from camisia foetus, and drawing blood from umbilical core, all of which can give accurate dignosis, but these methods are invasive technologys, which may cause injury of the fetal limbs and abortion. Many scholars have been looking for noninvasive methods to give prenatal diagnosis. Though the fact that fetal cells exsist in maternal peripheral blood has been testified by many scholars, the content of fetal cell is low. Another side, individual difference exsists and different duaration of pregnancy has different contents, so the fetal cells in maternal blood are delimited in clinic prenatal diagnosis. In 1997, scholar Lo[18]detected the special sequence on Y chromosome in maternal plasma, then we knew that free fetal DNA really exsists in maternal perepheral blood. Consequently, many other scientists[18]also detected fetal microsatellite polymorphism DNA sequence on chromosome 13,18,21 and paternal polymorphysm DNA sequence in maternal plasma using fluerecent quantitative PCR, so the limitation that detection only to male fetus was broken, pragmatic methods to give noninvasive prenatal diagnosis were eracted.We can extract fetal DNA through boiling and centrifuging method, this method was simple then before, we can avoid the sample polluting and DNA losing, Comparing to the method extracting chromosome DNA, boiling and centrifuging method is simpler, need no expensive reagent, but the DNA extracted using this method are mixed with much protein, we should purify the DNA with phenol and chloroform. We can remove the protein mixed with target DNA through magnetic beads enriching technology, so the interference by protein in PCR can be decreased, and the DNA losing in extraction and purification using phenol and chloroform can be avoided.Schorlar YanZiHe[24] has testified that the method to enrich little DNA fragments with magnetic beads has many merits. We can use the sticking and separating theory in magnetic field and improve efficiency in extracting DNA from plasma in high speed. This method is suitable especially to extracting little fragments, and the process can be repeated and avoid losing DNA.If we can extract the free DNA in maternal plasma with boiling and centrifuging method, then enrich the fetal DNA with immunology magnetic beads, the merits of the two methods can be used efficiently, the specificity and sensitivity can be improved.Object: To find a pragmatic method to enrich fetal DNA in maternal plasma making noninvasive prenatal diagnosis.Method: We have used magnetic beads covered with streptavidin to enrich fetal DNA. Firstly, we made probes labled with biotin hybridize with the free maternal plasma DNA to catch fetal fragments, then stuck these double strands by magnetic beads covered with streptavidin, discarded the supernatant in magnetic field and washed the magnetic beads, then denatured the double strand DNA and collected the fetal DNA in the magnetic field, then we used these fetal DNA to amplify the fetal special sequence on Y chromosome with 365 bp and 140 bp. After this, we have made the magnetic beads covered with streptavidin to save the cost of material. Firstly, we used EDC and NHS to activate the carboxy group, then added streptavidin to make the carboxy group bind to the amid. After covering the magnetic beads with streptavidin, we observed the dispersity of the beads through microscope and detected its special sticking fuction to make magnetic beads which are useful for enriching fetal DNA from maternal peripheral plasma.Result: ⑴In this study, we have used the magnetic beads to enrich the fetal DNA, and amplified the sequence in the DYS14 site on Y chromosome. In electrophoresis, we found the results are in accordance with the results directly using maternal plasma DNA.⑵we have detected the fragment with 365 bp in DYS14 region on Y chromosome, there are 16 positive results, after using the production in the first turn PCR tube to detect the 140 bp fragment of the 365 bp sequence, another 4 positive results were found. This nest polymerase chain reaction can improve the positive rate from 53.33% to 66.67%.Conclusion:The content of free fetal DNA enriched by immunology magnetic beads are enough for making PCR amplification, so this method can be used in detection of fetal DNA and prenatal diagnosis.