Optimization Of Multiple Displacement Amplification And Its Application In Single Cell

Multiple displacement amplification（MDA）is a widely used whole-genome amplification method,which has been used in various fields,including individual medical treatment,early diagnosis of tumor,archeology research,forensic testing and so on.This method of whole genome amplification（WGA）takes advantages of the high-fidelity phi29 DNA polymerase and 6N primers to synthetize the entire DNA template.The phi29 DNA polymerase has high strong strand displacement activity and proof-reading activity,leading to less biased amplification and higher genome coverage.In addition,the MDA process is carried out at room temperature,which is easily carried out.Although MDA technique has been successfully applied to many studies,there are several potential problems which may affect MDA results,including template input,Mg²⁺concentration,amplification time,reaction temperature and so forth,which may cause large amounts of nonspecific DNA amplification during MDA.In order to have a further understanding of MDA process,firstly,we analyzed several parameters which may influence the amplification;secondly,compared and optimized MDA protocol under different conditions of the template input,Mg²⁺concentration,amplification time and reaction temperature;thirdly,developed a MDA-WGA method based on above experiments applied to single-cell whole genome amplification.The main contents of this paper are as follows:1.We analyzed the yield of MDA under different conditions of the template input,Mg²⁺concentration,amplification time,reaction temperature based on other researches and obtained the influence curves of the five parameters.The results showed that template input（6-600 pg）has less effect on MDA results and the concentration of MDA products reached⁵00 ng/μL;the Mg²⁺concentration has a significant effect on MDA results,MDA showed the highest amplification efficiency when the Mg²⁺reached 10-15 mmol/L;the DNA yield increased with the extension of amplification time,and the quantity of the amplification reached a platform while the time was over 8 hours;the temperature has a close relationship with MDA yield,there was nearly no MDA products under condition of less than 20℃or more than 40℃,with the increase of temperature,the yield of MDA products increased dramatically,reaching highest concentration at about 30℃,and the analysis of next-generation sequencing showed that the amplification uniformity was much higher under 30-35℃than other temperatures.2.We constructed a procedure of MDA to applied to the single-cell whole genome amplification after comparising and summarizing different amplification parameters.The process of single-cell WGA is as follows:cell isolation,cell lysis and neutralization,MDA reaction.The YH-1 cell line was chosen as the research object to conduct multiple displacement amplification experiment.The effect of single-cell WGA was evaluated by housekeeping-gene PCR and coefficient of variation（CV）.The results of single-cell WGA showed that the procedure of MDA conducted in this paper is fit for single-cell WGA and shows good repeatability.The products can cover about 40%of the genome,and the relevant technical parameters are equivalent to the level of the single-cell genome sequencing research reported.