Short Tandem Repeat Genotyping Research Articles

Cover Picture: Electrophoresis 16'2010

AbstractIssue no. 17 is a regular issue comprising 18 manuscripts distributed over 5 separate parts. Part I has 7 research articles on some aspects of proteins and cell separations. Part II has 3 research articles on nucleic acid research including cloning/amplification, gene regulation and STR analysis. Part III offers ways of measuring diffusion and binding constants in two separate articles. Concentration and detection approaches are treated in 4 research articles making up Part IV. The last two articles in this issue (Part V) are on CEC and EKC describing a mixed mode monolithic stationary phase and a cyclodextrin‐modified MEEKC.Featured articles include:Lamp‐based wavelength‐resolved fluorescence detection for protein capillary electrophoresis: Set‐up and detector performance ((doi: 10.1002/elps.201000246))Electromigration diffusivity spectrometry: A way for simultaneous determination of diffusion coefficients from mixed samples ((doi: 10.1002/elps.201000252))Sample stacking capillary electrophoretic microdevice for highly sensitive mini Y short tandem repeat genotyping ((doi: 10.1002/elps.201000270))

Sample stacking capillary electrophoretic microdevice for highly sensitive mini Y short tandem repeat genotyping

Lab-on-a-chip provides an ideal platform for short tandem repeat (STR) genotyping due to its intrinsic low sample consumption, rapid analysis, and high-throughput capability. One of the challenges, however, in the forensic human identification on the microdevice is the detection sensitivity derived from the nanoliter volume sample handling. To overcome such a sensitivity issue, here we developed a sample stacking CE microdevice for mini Y STR genotyping. The mini Y STR includes redesigned primer sequences to generate smaller-sized PCR amplicons to enhance the PCR efficiency and the success rate for a low copy number and degraded DNA. The mini Y STR amplicons occupied in the 5- and 10-mm stacking microchannels are preconcentrated efficiently in a defined narrow region through the optimized sample stacking CE scheme, resulting in more than tenfold improved fluorescence peak intensities compared with that of a conventional cross-injection microcapillary electrophoresis method. Such signal enhancement allows us to successfully analyze the Y STR typing with only 25 pg of male genomic DNA, with high background of female genomic DNA, and with highly degraded male genomic DNA. The combination of the mini Y STR system with the novel sample stacking CE microdevice provides the highly sensitive Y STR typing on a chip, making it promising to perform high-performance on-site forensic human identification.

Microarray-based STR genotyping using RecA-mediated ligation

We describe a novel assay capable of accurately determining the length of short tandem repeat (STR) alleles. STR genotyping is achieved utilizing RecA-mediated ligation (RML), which combines the high fidelity of RecA-mediated homology searching with allele-specific ligation. RecA catalyzes the pairing of synthetic oligonucleotides with one strand of a double-stranded DNA target, in this case a PCR amplicon. Ligation occurs only when two adjacent oligonucleotides are base paired to the STR region without any overlap or gap. RecA activity is required to overcome the inherent difficulty of annealing repeated sequences in register. This assay is capable of determining STR genotypes of human samples, is easily adapted to high throughput or automated systems and can have widespread utility in diagnostic and forensic applications.

Molecular Analysis of Hydatidiform Moles

Distinction of hydatidiform moles from nonmolar specimens and subclassification of hydatidiform moles as complete hydatidiform mole (CHM), partial hydatidiform mole (PHM), or early CHM are important for both clinical practice and investigational studies. The risk of persistent gestational trophoblastic disease and hence, clinical management differs for CHMs, PHMs, and nonmolar specimens. However, diagnosis based solely on morphology suffers from poor interobserver reproducibility and remains problematic even for experienced gynecologic pathologists. The unique genetic features of CHMs (androgenetic diploidy), PHMs (diandric triploidy), and nonmolar specimens (biparental diploidy) allow for certain molecular techniques, including immunohistochemical analysis of p57 expression (a paternally imprinted maternally expressed gene) and molecular genotyping, to refine the diagnosis of hydatidiform moles. While p57 immunostaining alone can identify CHMs, which lack p57 expression due to the lack of maternal DNA, this analysis cannot distinguish PHMs from nonmolar specimens since both express p57 due to the presence of maternal DNA. Short tandem repeat genotyping, which can determine the parental source of polymorphic alleles, can distinguish among all of these entities by discerning androgenetic diploidy, diandric triploidy, and biparental diploidy to rigorously diagnose CHMs, PHMs, and nonmolar specimens, respectively. An algorithmic approach using these techniques to refine morphologic diagnosis has been developed for routine practice. This review discusses current issues in the diagnosis of hydatidiform moles, including the limitations of morphologic diagnosis, the need for refined diagnosis to assure accurate ascertainment of risk of persistent gestational trophoblastic disease associated with the different subtypes of hydatidiform moles, and the utility of ancillary immunohistochemical and molecular techniques for providing such refined diagnosis.

PopAffiliator: online calculator for individual affiliation to a major population group based on 17 autosomal short tandem repeat genotype profile

Because of their sensitivity and high level of discrimination, short tandem repeat (STR) maker systems are currently the method of choice in routine forensic casework and data banking, usually in multiplexes up to 15-17 loci. Constraints related to sample amount and quality, frequently encountered in forensic casework, will not allow to change this picture in the near future, notwithstanding the technological developments. In this study, we present a free online calculator named PopAffiliator ( http://cracs.fc.up.pt/popaffiliator ) for individual population affiliation in the three main population groups, Eurasian, East Asian and sub-Saharan African, based on genotype profiles for the common set of STRs used in forensics. This calculator performs affiliation based on a model constructed using machine learning techniques. The model was constructed using a data set of approximately fifteen thousand individuals collected for this work. The accuracy of individual population affiliation is approximately 86%, showing that the common set of STRs routinely used in forensics provide a considerable amount of information for population assignment, in addition to being excellent for individual identification.

Chromosome 21 non-disjunction and Down syndrome birth in an Indian cohort: analysis of incidence and aetiology from family linkage data

We analysed the family linkage data obtained from short tandem repeat (STR) genotyping of 212 unrelated Indian families having a single Down syndrome (DS) baby each, in order to explore the incidence and aetiology of this human aneuploidy in our cohort. The estimated values of maternal meiotic I and meiotic II non-disjunction (NDJ) errors of chromosome 21 (Ch 21) were approximately 78 and approximately 22%, respectively. Within the paternal outcome group, about 47 and 53% were accounted for NDJ at meiosis I and meiosis II, respectively. We estimated only approximately 2% post-zygotic mitotic errors. The comparison of average age of conception between controls and DS-bearing mothers revealed a significant difference (P<0.001) with DS-bearing women were on an average older than controls and meiotic II non-disjoined mothers were oldest among meiotic outcome groups. Our linkage analysis suggested an overall reduction in recombination by more than 50% on meiotic I non-disjoined maternal Ch 21 with error prone to susceptible chiasma formation within the approximately 5.1 kbp segment near the telomeric end. We stratified meiotic I non-disjoined women in three age groups, viz. young (<or=28 years), middle (29-34 years) and old (>or=35 years) and found linear decrease in the frequency of achiasmate meiosis from the young to the old group. In contrary, a linear increase in the multiple chiasma frequency from the young to the old group was observed. Considering these results together, we propose that the risk factors for Ch 21 NDJ are of two types, one being 'maternal age-independent' and the other being 'maternal age-dependent'. Moreover, a comparison of our present Indian dataset with that of other published data of ethnically different populations suggested that the genetics that underlies the NDJ of Ch 21 is probably universal irrespective of racial difference across human populations. The present study is the first population-based report on any DS cohort from the Indian subcontinent and our work will help future workers in understanding better the aetiology of this birth defect.

A sensitive method to extract DNA from biological traces present on ammunition for the purpose of genetic profiling

Exploring technological limits is a common practice in forensic DNA research. Reliable genetic profiling based on only a few cells isolated from trace material retrieved from a crime scene is nowadays more and more the rule rather than the exception. On many crime scenes, cartridges, bullets, and casings (jointly abbreviated as CBCs) are regularly found, and even after firing, these potentially carry trace amounts of biological material. Since 2003, the Forensic Laboratory for DNA Research is routinely involved in the forensic investigation of CBCs in the Netherlands. Reliable DNA profiles were frequently obtained from CBCs and used to match suspects, victims, or other crime scene-related DNA traces. In this paper, we describe the sensitive method developed by us to extract DNA from CBCs. Using PCR-based genotyping of autosomal short tandem repeats, we were able to obtain reliable and reproducible DNA profiles in 163 out of 616 criminal cases (26.5%) and in 283 out of 4,085 individual CBC items (6.9%) during the period January 2003–December 2009. We discuss practical aspects of the method and the sometimes unexpected effects of using cell lysis buffer on the subsequent investigation of striation patterns on CBCs.

Abstract 5040: Overcoming chemotherapy resistance via KISS1-mediated Cisplatin sensitization in HNSCC

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. While surgery and radiotherapy have traditionally been the modalities of choice for most disease sites in the head and neck, the integration of cisplatin (CDDP)-based systemic therapy has led to improvements in local and regional control of disease for patients with HNSCC. To elucidate the biological mechanisms underpinning cisplatin resistance in HNSCC, we developed a cell-based model of drug resistance by long-term exposure of tumor cell lines to escalating doses of CDDP over a 6-month period. We next tested the isogenic lineage of the OSC19CR (Cisplatin Resistant) cells by short tandem repeat genotyping (STR) and confirmed that by DNA genetic fingerprinting, the OSC19CR cells were identical to the parental OSC19 cell lines. We then utilized a flank model in mice to assess the in vivo biological behavior of the CDDP-resistant cells, and observed a signficant enhancement of both local tumor growth and distant metastasis in the OSC19CR cells, compared to the parental cells. Utilizing micro-CT imaging, we identified radiographically-visible pulmonary metastasis in the mice harboring the CDDP-resistant tumors, but not in the parental OSC19 mice. Further, in an orthotopic model of HNSCC, OSC19CR tumors were associated with adverse survival, compared to the parental OSC19 tumors. These findings suggested that CDDP resistance is associated with a biologically aggressive behavior, similar to that observed in human cancers. We next undertook a systems biology-based approach to elucidate the molecular mechanisms associated with the observed biological phenotype. Utilizing a high-throughput genomics-based approach, we identified a signficant decrease in KiSS1 expression in the OSC19CR cell line, compared to the parental cells. We further confirmed these observations utilizing end-point PCR, and also observed complete loss of KiSS1 at the protein level. Array CGH analysis identified no significant regions of genomic loss suggesting that transcriptional suppression may explain KiSS1 loss in CDDP resistant cell. To directly assess the role of KiSS1, the OSC19CR cells were stably transfected with a plasmid harboring the KiSS1 gene, which resulted in restoration of KiSS1 expression in the OSC19CR cells (OSC19CR-KiSS1). Wound scratch assays revealed that cellular migration was downregulated, but not completely abrogated, which suggested that the aggressive in vivo behavior may be due in part to the loss of KiSS1 in the OSC19CR cells. To confirm these findings either OSC19CR or OSC19CR-KiSS1 cells were inoculated into the tongues of nude mice. Survival studies revealed significant improvement in mouse viability in the OSC19CR-KiSS1 mice, with a rate that approximated the survivorship in the parental OSC19 cells. These studies provided further direct evidence for the role of KiSS1 in biologically aggressive HNSCC Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5040.

Abstract 3265: Immortalization and comprehensive characterization of a human ovarian adenocarcinoma cell line derived from an Ashkenazi Jewish patient

Abstract We have established and characterized a new ovarian cancer cell line derived from stage IV serous ovarian adenocarcinoma metastatic tissue from a 47 year old Ashkenazi Jewish patient. The patient developed ovarian cancer at age 44 and died of the disease at age 48. The tissue was obtained after an extensive secondary cytoreductive intraperitoneal surgery. Prior to the surgery the patient underwent primary cytoreduction followed by several systemic therapies with recurrences. A culture of the metastatic tissue was transfected with Simian Virus 40 Large T-Antigen and telomerase. The cell line has been in culture on and off during 3 years. The identity of the cell line was confirmed by Short Tandem Repeat genotyping on genomic DNA extracted from early and late passages as well as from frozen metastatic tissue. The cell line was further characterized by array-Comparative Genomic Hybridization and gene expression microarray; and by Spectral Karyotyping and Fluorescent In Situ Hybridization. The results of these analyses were compared, when appropriate, with those of frozen metastatic tissue. A low level of genetic instability was detected in the cell line, but the karyotypes seem to be near diploid and relatively stable, after several years of cultivation. We believe that this novel ovarian cancer cell line could contribute to the understanding of the molecular genetic basis of the disease in this advanced stage, and be representative of the tumors of a great number of patients that become resistant to the majority if not all known cancer therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3265.

Increasing the discrimination power of forensic STR testing by employing high-performance mass spectrometry, as illustrated in indigenous South African and Central Asian populations

Short tandem repeat (STR) typing has become the standard technique in forensic methodology for the identification of unknown samples. National DNA databases have been established that contain STR genotypes for intelligence purposes. Due to their success, national DNA databases have been growing so fast that the number of advantageous matches may become a logistic problem for the analysts. This is especially true for partial STR profiles as they display reduced discrimination power. To overcome this drawback, modified versions (so-called mini-STRs) of existing loci were introduced as well as new loci to improve the information content of (partial) STR profiles. We pursue an alternative approach that makes use of nucleotide variation within the amplified STR fragments, which can be discerned by mass spectrometry. We have developed an assay that determines molecular masses from crude STR amplicons which were purified and separated by a liquid chromatographic system directly hyphenated to an electrospray ionization mass spectrometer. We present here new population data of forensically relevant STRs in Khoisan and Yakut populations. These autochthonous groups were selected as they may harbor additional STR alleles that are rare or unobserved in modern humans from cosmopolitan areas, especially for the Khoisan, which are known to represent a very ancient human population. The analysis of the molecular mass of STRs offered a widened spectrum of allele variability escorted by enhanced forensic use. Thus, established STR data derived from fragment size analysis can still be used in casework or in the context of intelligence databasing.

Identification of an avirulent Entamoeba histolytica strain with unique tRNA-linked short tandem repeat markers

Highly polymorphic, non-coding short tandem repeats (STR) are scattered between the tRNA genes in Entamoeba histolytica in a unique tandemly arrayed organization. STR markers that correlate with the virulence of individual E. histolytica strains have recently been reported. Here we evaluated the usefulness of tRNA-linked STR loci as genetic markers in identifying virulent and avirulent strains of E. histolytica from 37 Japanese E. histolytica samples (12 diarrheic/dysenteric, 20 amebic liver abscess (ALA), and 5 asymptomatic cases). Twenty three genotypes, assigned by combining the STR sequence types from all 6 STR loci, were identified. One to 8 new STR sequence types per locus were also discovered. Genotypes found in asymptomatic isolates were highly polymorphic (4 out of 5 genotypes were unique to this group), while in symptomatic isolates, almost half of the genotypes were shared between diarrhea/dysentery and ALA. One asymptomatic isolate (KU27) showed unique STR patterns in 4 loci. This strain, though associated with the typical pathogenic zymodeme II, failed to induce amebic liver abscess by animal challenge, which suggests that inherently avirulent E. histolytica strains exist, that are associated with unique genotypes. Furthermore, STR genotyping and in vivo challenge of 2 other asymptomatic isolates (KU14 and KU26) verified the covert virulence of these strains.

Molecular Genotyping of Hydatidiform Moles: Analytic Validation of a Multiplex Short Tandem Repeat Assay

Distinction of hydatidiform moles from non-molar (NM) specimens, as well as their subclassification as complete (CHM) versus partial hydatidiform moles (PHM), is important for clinical management and accurate risk assessment for persistent gestational trophoblastic disease. Because diagnosis of hydatidiform moles based solely on morphology suffers from poor interobserver reproducibility, a variety of ancillary techniques have been developed to improve diagnosis. Immunohistochemical assessment of the paternally imprinted, maternally expressed p57 gene can identify CHMs (androgenetic diploidy) by their lack of p57 expression, but cannot distinguish PHMs (diandric monogynic triploidy) from NMs (biparental diploidy). Short tandem repeat genotyping can identify the parental source of polymorphic alleles and thus discern androgenetic diploidy, diandric triploidy, and biparental diploidy, which allows for specific diagnosis of CHMs, PHMs, and NMs, respectively. In this study, a retrospectively collected set of morphologically typical CHMs (n = 8), PHMs (n = 10), and NMs (n = 12) was subjected to an analytic validation study of both short tandem repeat genotyping and p57 immunohistochemistry. Several technical and biological problems resulted in data that were difficult to interpret. To avoid these pitfalls, we have developed an algorithm with quantitative guidelines for the interpretation of short tandem repeat genotyping data.

An integrated microdevice for high‐performance short tandem repeat genotyping

Short tandem repeat (STR) analysis provides genetic fingerprinting of individuals, and is considered as a powerful and indispensable technique for forensic human identification. However, the current state-of-the-art STR genotyping processes and instruments are labor intensive, expensive, time consuming, and lack portability. Micro-total-analysis systems or lab-on-a-chip platforms based on microfabrication technologies have the capability to miniaturize and integrate bioanalysis steps in a single format. Recent progress in microsystems has demonstrated their successful performance for the forensic STR typing with a reduced cost, high speed, and improved high throughput. The purpose of this review article is to highlight up-to-date work on advanced microdevices for high-throughput STR genotyping, and a portable integrated microsystem for on-site forensic DNA analysis.

STR genotyping of DNA extracted from used Triage kits

This study examined whether short tandem repeat (STR) genotyping can be performed using DNA remaining in Triage kits used to screen for drugs of abuse in urine. STR genotyping was successful for 15 loci using 12 kits stored for 1–6 months at room temperature. These results suggest that STR genotyping for human identification can be performed using DNA extracted from used Triage kits.

Paternity testing involving human remains identification and putative half sister: Usefulness of an X-hexaplex STR markers

Paternity testing is being increasingly requested with the aim of challenging presumptive fatherhood. The ability to establish the biological father or mother is usually based on the genotyping of autosomal short tandem repeats (STR). However, in the last few years the use of X chromosomal STR (chr-XSTRs) markers gained strong interest in paternity involving females and specially in kinship analysis where the alleged father is absent since true daughters share the single X-chromosome from their biological father. In this work, we present an approach that combines data from fifteen autosomal and six chr-X STRs in order to know the DNA profiles of presumed relatives by genotyping human remains and/or investigate female as putative half sister or unrelated. As a result, the LR over all available loci for two cases were 1,432,331,424 and 9,357 corresponding to probabilities of 99.9999999% and 99.989%, respectively. The LR values associated to the high probabilities confirm the usefulness of the X-hexaplex STR markers in resolving relationships even thought genotyping human remains.

Linkage of a Mild Late-Onset Phenotype of Fuchs Corneal Dystrophy to a Novel Locus at 5q33.1-q35.2

To identify the disease locus associated with autosomal dominant Fuchs corneal dystrophy (FCD) in a large family and to compare the progression of severity in families mapped to the FCD1 and FCD2 loci. Seventeen individuals in a large family were examined by slit lamp biomicroscopy. Blood samples were collected, DNA was extracted, and a genome-wide scan was performed with a microarray SNP chip. After initial generation of a genome-wide, two-point LOD score, linkage was confirmed and the critical interval was established by genotyping of short tandem repeat (STR) microsatellite markers. A genome-wide linkage scan localized the disease interval to the long arm of chromosome 5, with a maximum two-point parametric LOD score of 3.41. Haplotype analyses refined the critical interval to 5q33.1-q35.2, spanning a 27-Mb (29-cM) region. Clinical examination of affected individuals in this family revealed an early onset of FCD at approximately age 40, after which progression of the disease was significantly attenuated compared to the FCD1- and FCD2-linked families. Late-onset FCD is linked to a novel locus on 5q33.1-q35.2 and is associated with a milder severity in age at onset and rate of progression than the FCD1 and FCD2 loci. Correlation of individual genotypes with unique rates of disease progression will provide important tools for disease management, as well as for identifying the underlying genetic lesion, offer insight into the pathomechanism of FCD.

Pure monosomy and pure trisomy of 13q21.2–31.1 consequent to a familial insertional translocation: Exclusion of PCDH9 as the responsible gene for autosomal dominant auditory neuropathy (AUNA1)

Insertional translocations (IT) are rare structural rearrangements. Offspring of IT balanced carriers are at high risk to have either pure partial trisomy or monosomy for the inserted segment as manifested by "pure" phenotypes. We describe an IT between chromosomes 3 and 13 segregating in a three-generation pedigree. Short tandem repeat (STR) segregation analysis and array-comparative genomic hybridization were used to define the IT as a 25.1 Mb segment spanning 13q21.2-q31.1. The phenotype of pure monosomy included deafness, duodenal stenosis, developmental and growth delay, vertebral anomalies, and facial dysmorphisms; the trisomy was manifested by only minor dysmorphisms. As the AUNA1 deafness locus on 13q14-21 overlaps the IT in the PCDH9 (protocadherin-9) gene region, PCDH9 was investigated as a candidate gene for deafness in both families. Genotyping of STRs and single nucleotide polymorphisms defined the AUNA1 breakpoint as 35 kb 5' to PCDH9, with a 2.4 Mb area of overlap with the IT. DNA sequencing of coding regions in the AUNA1 family and in the retained homologue chromosome in the monosomic patient revealed no mutations. We conclude that AUNA1 deafness does not share a common etiology with deafness associated with monosomy 13q21.2-q31.3; deafness may result from monosomy of PCHD9 or another gene in the IT, as has been demonstrated in contiguous gene deletion syndromes. Precise characterization of the breakpoints of the translocated region is useful to identify which genes may be contributing to the phenotype, either through haploinsufficiency or extra dosage effects, in order to define genotype-phenotype correlations.

Identification using DNA from skin contact: Case reports

The DNA content of different types of forensic materials can vary substantially. “Trace DNA” is the minute quantity of DNA transferred through skin contact. Here, we report three cases of identification of trace DNA using conventional short tandem repeat (STR) or mitochondrial DNA (mtDNA). DNA was successfully obtained from fabrics by swabbing or by direct extraction and subjected to STR genotyping or mtDNA typing. In two cases, there was no amplification of PCR products containing the STR loci. This indicates that the areas chosen for DNA extraction contained trace DNA and DNA from more than one source. Therefore, it is important for forensic investigators performing DNA typing to know where an item has been frequently touched by victims and/or offenders as this will influence the choice of sites on the item to be used for DNA extraction.

Gene diagnosis for nine Chinese patients with DMD/BMD by multiplex ligation‐dependent probe amplification and prenatal diagnosis for one of them

This study aims to perform gene diagnosis for nine patients with Duchenne/Becker muscular dystrophy (DMD/BMD) and their parents with multiplex ligation-dependent probe amplification (MLPA), and to carry out prenatal gene diagnosis for one of them. Genomic DNA of the peripheral blood and fetal amniotic fluid cell was extracted from the pedigrees' members with DMD/BMD. Gene diagnosis was performed for theses pedigrees' members using a SALSA KIT. Short tandem repeats (STR) genotyping and X-linkage analysis were performed for the pedigree members of the fetus, which was used in the prenatal diagnosis. MLPA analysis results show that five of nine patients (DMD-1, DMD-2, DMD-4, DMD-8, and DMD-9) with DMD/BMD were found to have several hemizygous exon deletions in the dystrophin gene. The other patients and the fetus did not have any hemizygous deletion or duplication of any exons. The genomic DNA of the fetus was not contaminated by his mother's DNA as identified by STR genotyping. In addition, X-linkage analysis results show that the only X chromosome of the fetus comes from one of his mother's normal X chromosomes. Combined with STR genotyping and X-linkage analysis, MLPA is a convenient, highly effective and reliable gene diagnosis technique for congenital genetic disease.

Single nucleotide polymorphism genotyping by mini-primer allele-specific amplification with universal reporter primers for identification of degraded DNA

Single nucleotide polymorphism (SNP) is informative for human identification, and much shorter regions are targeted in analysis of biallelic SNP compared with highly polymorphic short tandem repeat (STR). Therefore, SNP genotyping is expected to be more sensitive than STR genotyping of degraded human DNA. To achieve simple, economical, and sensitive SNP genotyping for identification of degraded human DNA, we developed 18 loci for a SNP genotyping technique based on the mini-primer allele-specific amplification (ASA) combined with universal reporter primers (URP). The URP/ASA-based genotyping consisted of two amplifications followed by detection using capillary electrophoresis. The sizes of the target genome fragments ranged from 40 to 67 bp in length. In the Japanese population, the frequencies of minor alleles of 18 SNPs ranged from 0.36 to 0.50, and these SNPs are informative for identification. The success rate of SNP genotyping was much higher than that of STR genotyping of artificially degraded DNA. Moreover, we applied this genotyping method to case samples and showed successful SNP genotyping of severely degraded DNA from a 4-year buffered formalin-fixed tissue sample for human identification.