Application Of HRM In Genotyping Of Genetic Markers From Forensic Challenging Biological Samples

Objective:High-resolution melting(HRM)analysis is a versatile method for variant scanning and genotyping.It involves amplification of the target in the presence of a saturation dye by the polymerase chain reaction(PCR).HRM analysis can be performed in one closed-tube,which does not require additional post-PCR separations and greatly reduces the possibility of contamination.HRM is faster,simpler,and less expensive than alternative approaches requiring labeled probes.Considering the many advantages of HRM analysis,many researchers have tried to apply this method to forensic research.This paper intends to summarize the principle,technical characteristics,limitations and application of HRM analysis in forensic science.This study intends to use HRM analysis to analyze genetic markers of trace or degraded samples in order to obtain forensic-related individual information.Degraded and low-copy-number(LCN)DNA samples are commonchallenging materials in forensic casework because they increase the difficulty of sample processing and reduce the possibility of obtaining genetic information from DNA.High-resolution melting(HRM)curve analysis is promising for genotyping genetic markers and has been applied to the detection of LCN and degraded DNA in the field of forensic science.However,the exact assessment based on HRM at multiple genetic markers from both degraded and LCN DNA materials has not been optimized.To explore the ability of HRM to genotype LCN and degraded DNA samples,we selected three genetic markers to genotype in experimental LCN and degraded DNA and practical hair shaft materials,which are often encountered as degraded and LCN DNA in forensic medicine.The results show that DNA samples of as low as 100 pg and as short as 60 bp were successfully genotyped by the HRM assay at all three genetic markers,whereas in hair shaft DNA,two loci were accurately genotyped.The HRM assay established in this study can be applied to LCN and degraded DNA analysis in forensic casework and can act as a reference point before genotyping short tandem repeat markers.Developing the HRM strategy for genotyping DNA genetic markers enriches detectable targets in hair shaft samples and provides valuable data for further exploration.Methods:1.This study designed short fragment amplification specific primers for c.261delG and c.297A>G on exon 6 and c.803G>C,c.1009A>G and c.1061delC,on exon 7 in ABO gene,a 3bp insertion in the AMELY gene for HRM analysis.2.DNA samples which were extracted from whole blood using the sodium dodecyl sulfate-proteinase K phenol-chloroform method.3.Hair root was used for DNA extraction according to the manufacturer’s recommended procedure for the PrepFiler~?Forensic DNA Extraction Kit(Thermo Fisher Scientific).4.The next 2 cm of each hair shaft was cut in small fragments,and DNA was extracted using DNeasy~?Blood&Tissue Kit(QIAGEN)and PrepFiler~?Forensic DNA Extraction Kit(Thermo Fisher Scientific).5.Artificially degraded DNAs were prepared from 8 genomic samples by DNase I digestion.6.Combine the results of PCR amplification and touch down PCR between 65°C and 53°C on LightCycler~?480 to select the annealing temperature to optimize HRM conditions.7.HRM data were analysed,normalized,and temperature-shifted by the LightCycler~?480 GeneScanning software version 1.5 and then converted to a derivative plot.Cp values and PCR efficiency were estimated using LightCycler?480 Abs Quant/2nd Derivative Max Software.Results:1.Based on the melting curve patterns,peaks,heights,and Tm values,the genotypes of all 4 loci in the ABO gene could be simultaneously and accurately distinguished.2.This study allowed the identification of A,A201,A205,B,O01,and O02 alleles in the ABO gene.Consequently,a total of 14 genotypes were identified by this method:A/A,A/O01,A/O02,A201/O01,A205/O01,B/B,B/O01,B/O02,A/B,A201/B,A205/B,O01/O01,O02/O02,O01/O02.The fusion protein hZP3-6His was purified by affinity chromatography.3.For all three genetic loci,accurate and reproducible results were obtained down to 100 pg DNA.4.When DNA fragments were over 60 bp in length,stable and reliable results were obtained at all three loci by HRM analysis.However,when the DNA fragments were shorter than 60 bp,only c.261delG could still be genotyped stably in all samples.5.For c.803G>C locus and the 3-bp GAT insertion in AMELY,the genotypes were obtained accurately and repeatedly under some modifications,for the c.261delG locus,the genotyping was failed.Conclusion:1.HRM assay presented here is a reliable and rapid method for ABO genotyping at c.261delG,c.297A>G,c.1009A>G,and c.1061delC in the ABO gene.Both individual and multiplexed HRM curves were distinguishable among all genotypes.2.The established HRM assay in this study may be applied to LCN and degraded DNA analyses in forensic casework,and it can act as a reference point before typing STR markers.