Generating DNA profile from low copy number DNA: Strategies and associated risks

Many shreds of evidence found on the crime scenes contain a trace amount of DNA which results in insignificant profiling results for subsequent comparison. This can nullify the potential evidence material and hamper investigation process. Over the years, different strategies have been employed by various DNA testing laboratories to create interpretable DNA profiles generated from low template of DNA. This review highlights different strategies used by forensic laboratories worldwide for creating complete DNA profiles from low copy number template for comparison purposes along with its associated risks for forensic purposes.

The detectable time limit of intact STR profile for human tissues in unbuffered formalin / 中国法医学杂志

Objective To evaluate the STR profiling availability of formalin-fixed tissues and the detectable time limit of intact STR profile.Methods The different human tissues were fixed with 10-fold diluted commercial 40%formalin fixative for different duration under 15～20℃,and then DNA was extracted using the method of QIAamp~(R)DNA and IQ~(TM) DNA System.The extracted DNA was quantified with QuantifilerTM kit and amplified by both AmpFSTR identifiler kit and AmpFSTR MiniFiler kit.The STR profile was analyzed by GeneMapper ID v3.2 on 3100-Avant.Resuls The STR profiling availability of formalin-fixed tissues was relevant to the formalin fixing duration mainly.as well as the type of tissues and the template concentration and protocol of DNA extracting.The optimal ranges of template concentration is 1～3ng/μL and the QIAamp extracting method was preferable.There are differences in the degradation rate between various types of tissues in the unbuffered formalin fixative,and the lung tissue showed the slowest rate and liver and intestine tissues the fastest.Intact miniSTR profile of all the tissues detected could be obtained within 15 days duration of formalin fixing while intact STR profile could be obtained within 4 days.Conclusion The major factor that impact the availability of STR profiling of formalin-fixed tissues is the fixing duration in unbuffered formalin,as well as the type of tissues,method of extraction,concentration of PCR template and the kinds of STR loci.

STR profiling on authentication of human cell lines in cell banks / 中华微生物学和免疫学杂志

Objective To study the profiling and authentication of human cell lines in cell bank of our department using short tandem repeat (STR) loci and to analyze the situation of cell contamination and misjudgment. Methods Sixty-one human cells including cells collected and preserved by our cell bank and cells from the other departments were detected by the 16 STR loci-method. To analyze the cross contamina-tion between human cell lines, the results were aligned with profiles published by international cell banks. Isoenzyme detection was employed to authenticate the cell species when the STR signal can not be detected. Results Among the 61 cells, specific profiles were produced by 41 ceils and there was no cross contamina-tion. Thirty-six cells had the completely same STR profiles in 9 STR loci with the same cells preserved by ATCC or JCRB, while 5 cells have different profiling just in the vWA loci. The cells referred above can be recognized as correct cells; Eleven cells (18.0%) were the false cells. Among them, cancer cells of tongue named Tca8113 and cancer cell of liver named HHCC(changed to FHCC98 now) had the same profile with HeLa and HeLa S3 respectively; Two ceils both named HUT-102 have the completely different profiles with ATCC; The signal of 4 cells was not be detected, and all of them were determined as hamster cell lines by u-sing isoenzyme detection. Also 2 cells were identified to be mixed cells. Conclusion The phenomenon of cell misjudgment and cross contamination between cells is serious. Authentication of cell lines correctly, es-pecially for the re-authenticatian of domestic self-established cells, is very important for the guarantee of the reliability and reproducibility for scientific researches.

STR profiling on authentication of human cell lines for production / 中华微生物学和免疫学杂志

Objective To study the application of short tandem repeat (STR) profiling in quality control of human cell lines used for biological production. Methods The methods detecting 9 and 16 STR loci to identify human cell lines by PCR-capiilary electrophoresis were established respectively. Human cell lines, which were derived from many corporations and including diploid cell strains used for virus-vaccine production and 293 cell lines used for gene therapy products, were analyzed and compared by these two methods. Results The STR profiling methods used for authentication of human cell lines were established. Most of human diploid cell strains(20/21 ) used for virus-vaccine production from 13 corporations were iden-tiffed as the intended cells and no cross-contamination was found. However, one MRC-5 cells was identified as a false cell line and one MRC-5 had 3 alleles in D13S317 locus. For 12 strains of 293 cell lines, there were significant differences in STR profiling from different manufactures, which was likely be explained that the sources and gene modifications of these 293 cell lines are not well known and their genes are unstable during passage. Conclusion The STR profiling method has the advantages of high sensitivity and specifici-ty, and can be used for authentication of each of human cell lines for biological production.