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CN-107760773-B - Method for carrying out scRRBS analysis on embryo culture solution

CN107760773BCN 107760773 BCN107760773 BCN 107760773BCN-107760773-B

Abstract

The present invention provides a method for Single cell simplified representative bisulfite sequencing (Single-cell reduced-representation bifulfite sequencing, scRRBS) of embryo culture fluid. The invention adopts medical waste (blastula culture solution) in 'test-tube infant' operation as raw material, can simultaneously perform double analysis on chromosome aneuploidy condition and DNA methylation condition of embryo, evaluate development potential of embryo from the brand new angle of epigenetic and reaction of embryo and culture environment, provide new reference for selecting 'correct' embryo in auxiliary reproduction, and provide strong support for improving success rate of 'test-tube infant' period.

Inventors

  • FENG TAO
  • FEI JIA
  • ZHANG GUIRONG
  • JIN ZHIPING

Assignees

  • 北京中仪康卫医疗器械有限公司

Dates

Publication Date
20260505
Application Date
20171026

Claims (14)

  1. 1. A method for single-cell simplified representative bisulfite sequencing (scRRBS) of embryo culture fluids for non-diagnostic purposes, comprising the steps of: (1) Obtaining a noninvasive sample, obtaining fertilized eggs through a single sperm intracytoplasmic injection (ICSI), culturing the fertilized eggs in a blastomere culture medium until the blastomere period, transferring the fertilized eggs into a blastomere culture medium for culturing until the blastomere maturation period, manually shrinking the blastocysts to discharge liquid in a blastocyst cavity, and completely transferring the culture liquid into a PCR tube for standby; (2) Constructing scRRBS sequencing libraries of embryo culture solutions, namely, subjecting the culture solution sample in the step (1) to cleavage by a lyase, mspI enzyme digestion of a cleavage product, end repair and addition A, joint connection and joint ring opening treatment, CT conversion treatment and purification recovery, PCR amplification, purification and secondary amplification, and recovery of a target product; (3) Sequencing the scRRBS sequencing library constructed in the step (2); (4) The double analysis for non-diagnosis purpose comprises analyzing the obtained data by adopting a methylation analysis flow to obtain the DNA methylation state of the sample, and analyzing the chromosome aneuploidy state of the sample by adopting a general PGS analysis flow; Wherein in step (2), in the Msp I cleavage, the amount of unmethylated lambda DNA as a reference DNA is about 1% of the amount of DNA in the sample, the adaptor ligation uses a methylated adaptor, the adaptor is ligated with T4 DNA ligase, the adaptor is pre-ligated for 30min at 16 ℃, then the ligation is performed overnight or not less than 8 hours at 4 ℃, the adaptor is inactivated by a treatment at 50-75 ℃ for 20min after the ligation is completed, the adaptor is a hairpin sequence, there is one U base in the hairpin loop structure, and the adaptor loop structure is opened by a treatment at 37 ℃ for 20-50min with USER enzyme.
  2. 2. The method according to claim 1, wherein in the step (1), fertilized eggs are obtained by selecting qualified mature egg cells in MII stage, thoroughly removing the granular cells attached to the outside of the egg cells as much as possible, selecting healthy sperm, and obtaining fertilized eggs by single sperm injection.
  3. 3. The method according to claim 1, wherein in step (1), after culturing fertilized eggs in G1 medium to blastomere phase, embryos are washed several times in blastomere medium to remove possible residual granulosa cells, and then transferred into 5-50. Mu.l fresh blastomere medium droplets, each of which cultures one embryo.
  4. 4. The method according to claim 1, wherein in the cleavage with the cleavage buffer in step (2), the cleavage enzyme is selected from one or more of proteinase K, qiagen Protease, pepsin, papain, trypsin and lysozyme at an enzyme concentration of 1-30 μg/ml, and the cleavage buffer comprises 2-200mM Tris-EDTA, 1-50mM KCl, 0.1-5 wt% of a surfactant selected from one or more of Triton X-100, SDS, tween-20, NP 40.
  5. 5. The method according to claim 4, wherein the temperature of incubation of the lyase is 37-65℃for 30min to 12h, the inactivation temperature is not more than 80℃and the time is 10-45min.
  6. 6. The method of claim 1, wherein in the Msp I cleavage of step (2), the buffer is Tango buffer, the cleavage temperature is 37 ℃ for 1-6 hours, the inactivation temperature is 60-80 ℃ for 10-30 minutes.
  7. 7. The method of claim 1, wherein the end repair and addition a of step (2) uses DNA polymerase Klenow fragment, the buffer is Tango buffer, the reaction temperature is 37 ℃, the repair time is 20-60min, and the inactivation time is 60-80 ℃ for 5-30min.
  8. 8. The method of claim 1, wherein the CT conversion is performed in step (2) using bisulfite.
  9. 9. The method of claim 1, wherein the PCR amplification of step (2) uses one or both of KAPA hifu+master Mix, phusion high-fidelity (HF) PCR MASTER Mix with HF buffer.
  10. 10. The method according to claim 1, wherein the PCR amplification procedure is 98℃for 2min, 98℃for 20s,60℃for 30s,72℃for 60s,6-12 cycles, 72℃for 5min, 4℃for preservation, and the second amplification procedure is 98℃for 2min, 98℃for 20s,60℃for 30s,72℃for 60s,15-21 cycles, 72℃for 5min, and 4 ℃.
  11. 11. The method of claim 10, wherein after completion of PCR amplification, the amplified product is purified by 0.5-1.5 volume XP magnetic beads.
  12. 12. The method according to claim 1, wherein the target DNA fragment of 200-700bp is recovered in step (2).
  13. 13. The method of claim 12, wherein the gel is recovered using 1.5-2% tae or TBE agarose electrophoresis.
  14. 14. The method of claim 1, wherein NGS sequencing is used in step (3).

Description

Method for carrying out scRRBS analysis on embryo culture solution Technical Field The invention relates to the fields of biomedicine and molecular cell biology, in particular to a method for performing Single-cell simplified representative bisulfite sequencing (Single-cell reduced-representation bifulfite sequencing, scRRBS) on embryo culture fluid, which utilizes epigenetic analysis technology to analyze embryo chromosome state and DNA methylation state on the embryo culture fluid. Background Since 7 months in 1978, the first example of "test tube baby" was created by Lobert G. Edwards, the pioneer in 2010, which acquired Nobel medical and physiological rewards, millions of test tube babies were created worldwide, and the term "test tube baby" was well known. Although the technology of test tube infants has undergone the first and second generations with the rapid development of the technology, the technology has come into the third generation nowadays, but the overall success rate still does not reach the expectations of people, and the success rate is generally about 40% at present. The technique of test tube infants involves multiple disciplines such as obstetrics, gynecology, reproductive physiology, genetics, embryology and developmental biology, and also relies on laboratory techniques and clinical disciplines. A test tube infant cycle comprises comprehensive inspection of parents, ovulation promotion to obtain healthy ova, sperm acquisition, in vitro fertilization, fertilized ova and embryo culture, proper embryo transplantation process selection, prenatal series monitoring inspection after transplantation and the like until the healthy infant is born. In such a period of up to about one year, many factors affect the developmental potential of the embryo, which is a significant reason for the success of this period. The main standard for embryo development potential evaluation is a morphological standard, which is not uniform but accepted by the vast medical staff and patients. With the development of technology and the depth of clinical analysis, researchers have found that the main cause of most abortions is chromosomal aneuploidy of embryos. Morphologically normal embryos do not necessarily have normal chromosomal aneuploidy, which is a significant cause of post-implantation failure. Various aneuploidy screens (PGS) have been developed that select for normal chromosomal embryo transfer by chromosome screening of a portion of the cells of the embryo to increase success rate. However, there is still a "normal" failure after good embryo transfer following PGS techniques. This is because, after in vitro fertilization of assisted reproduction, the embryo is cultured in vitro for 3 to 5 days, and during this period, the embryo development is severely affected by the culture environment, since the period from fertilized eggs to morula or blastocyst stage is performed under non-maternal conditions. In the early stage of embryo development, the epigenetic status of the embryo changes drastically, and the change may be affected by the external environment to change, thereby affecting the development potential of the embryo, and causing some morphologically high-quality embryos with normal chromosome states to decrease the development potential due to the interference of epigenetic inheritance and cause failure after transplantation. In the current preimplantation screening, however, no analysis of the epigenetic status of the embryo was performed. Meanwhile, currently, the mainstream PGS adopts trophoblast cell biopsy at the blastocyst stage, and although the biopsy has not proved to be harmful to embryos and test-tube infants, the operation technology is high, and the embryo is stopped due to improper operation. CN105368936a uses the waste liquid after embryo culture as material to try to achieve noninvasive detection, but still only analyzes the chromosome status. In summary, the current mainstream techniques for evaluating embryo development potential have the following disadvantages: biopsy samples have high technical requirements on embryo handling, and even if the operation is good, certain damage can be caused to the embryo, if the operation is wrong, the embryo is likely to be injured, and serious consequences are caused Trophoblast cells may be chimeric and a small number of 3-5 cells may not be fully representative of an embryo, thus leading to misjudgment. Evaluation from chromosome status alone, ignoring epigenetic changes important in the early embryo phase, does not provide a more comprehensive reference for evaluating embryo developmental potential. Thus, there is an urgent need for a new method of detecting embryo developmental potential that is non-invasive and simultaneously provides multiple angle references. Disclosure of Invention The invention improves the existing detection technology completely, introduces an epigenetic analysis method, utilizes the blastula culture solution to ana