A Study On The Effect Of MALBAC Whole-genome Amplification On LCN-DNA STR Typing

Background The advantages such as wide range of distribution and high degree of genetic polymorphism made Short tandem repeats(STRs)become important genetic markers used by forensic science laboratories in many countries.Currently,mature commercial kits of each category can simultaneously amplify more than a dozen gene loci.However,for most conventional STR typing kits,1 ng is the ideal initial template volume.In the daily practice of forensic medicine,it is sometimes encountered that the quality of the samples is not ideal.These samples often have unfavorable or even negative typing results when they are typed using conventional kits.The ever-evolving forensic DNA analysis technology is changing with each passing day,and the range of detection content is also expanding.Low-copy template biological sample correlation analysis technology has received more and more attention,and the application demand for LCN-DNA STR typing has also increased accordingly.Under limited conditions,scholars have come up with many different methods to get the ideal STR pattern.Such as increasing the number of PCR cycles,changing PCR reaction parameters,purifying amplification products,and whole genome amplification techniques,each method has its advantages and disadvantages,and the results of typing have also been improved to varying degrees.Among them,the whole genome amplification technology is the non-selective amplification of all the genome sequences.The main purpose of the whole genome amplification technology is to increase the total amount of DNA and prevent the occurrence of sequence biases.With the entire surface of the template genome,its amplification products can provide sufficient template for subsequent gene amplification or sequencing analysis.MALBAC is a newly proposed technique for genome-wide amplification in recent years.It combines the characteristics of MDA method and PCR method,showing higher balance and specificity than MDA method,and lower allele loss rate.Here,we investigate the effect of MALBAC whole genome amplification technique on LCN-DNA STR typing results,including product yield and alleles.Object Explore the feasibility of MALBAC application in forensic LCN-DNA STR analysis by comparing the yield and typing results of the MALBAC whole genome amplification technique with the use of very low template amounts of amplified products.Methods(1)Select blood samples of 5 unrelated healthy Han individuals in Wuhan,Hubei Province and use the QIAamp~?DNA Blood Mini kit to extract genomic DNA.(2)Quantify the extracted genomic DNA using the Quantifiler~?Trio DNA Quantification Kit.(3)Each quantitative sample was respectively released at 100 pg/μL,60 pg/μL,40 pg/μL,20pg/μL,and 10 pg/μL,(4)Whole-genome amplification of the diluted sample using the MALBAC?single-cell whole genome amplification kit.(5)Using the Nanodrop 2000 ultra-micro spectrophotometer to quantify the products after whole genome amplification.(6)Use the AGCU Expressmarker 22 Fluorescence Detection Kit to perform capillary electrophoresis detection typing on the whole genome amplification product and record the classification data and analyze it.Result(1)MALBAC genome-wide amplification technology has a significant effect in increasing the amount of DNA template,and the final product yield is 1-6μg.(2)MALBAC whole genome amplification technology significantly improved the STR genotyping success rate of samples with very low template amounts.Conclusion The MALBAC whole genome amplification technology has significantly improved the LCN-DNA STR typing in both product quantity and typing results,while maintaining good allele coverage and sensitivity.The application lay the foundation for subsequent application in forensic LCN-DNA STR research.