Amplified Product Length Polymorphism Research Articles

Mitochondrial haplogrouping of the ancient brown bears (Ursus arctos) in Bulgaria, revealed by the APLP method

In order to investigate the detailed zoogeographical history of brown bears (Ursus arctos) in Bulgaria and their relationships with populations in neighboring regions of Europe and Asia, the amplified product length polymorphism (APLP) method for the mitochondrial DNA (mtDNA) haplogrouping was applied to ancient bone remains. The results showed that haplogroups for 12 of 31 samples (38.7%) were successfully classified using the APLP method, although partial sequences of the mtDNA control region were determined with PCR product-direct sequencing for only 6 samples (19.4%). Even among 25 samples, of which nucleotide sequences could not be determined, the APLP method successfully classified haplogroups of 6 samples (6/25, 24.0%), indicating an advantage of the APLP method. In Bulgaria, although both the Balkan/Italian lineage of mtDNA (clade 1b) and the Eastern European lineage (clade 3a1) have been identified from modern bears as reported in other studies, all bear remains examined in the present study had APLP haplogroup W. The mtDNA phylogenetic analysis showed that the ancient Bulgarian brown bears had clade 1b. This indicates that clade 1b was originally distributed in Bulgaria, whereas those with clade 3a1 could have entered from Romania to Bulgaria. In addition, the APLP and phylogenetic analysis of recent skin samples from Turkey showed that they have mtDNA of the Middle Eastern/Turkish lineage (referred to clade 7a). Therefore, Bulgaria on the Balkan Peninsula might have been located between two distribution borders: one is between clades 1b and 3a1, and the other is between clades 1b and 7.

Continental Origin for Q Haplogroup Patrilineages in Argentina and Paraguay.

Haplogroup Q originated in Eurasia around 30,000 years ago. It is present in Y-chromosomes from Asia and Europe at rather low frequencies. Since America is undoubtedly one of the continents where this haplogroup is highly represented, it has been defined as one of the founding haplogroups. Its M3 clade has been early described as the most frequent, with pan-American representation. However, it was also possible to find several other haplogroup Q clades at low frequencies. Numerous mutations have been described for haplogroup Q, allowing analysis of its variability and assignment of its geographic origin. We have analyzed 442 samples of unrelated men from Argentina and Paraguay belonging to haplogroup Q; here we report specifically on 27 Q (xM3) lineages. We tested 3 single-nucleotide polymorphisms (SNPs) by amplified product-length polymorphism (APLP) analysis, 3 SNPs for restriction fragment length polymorphism (RFLP) analysis, 15 SNPs by Sanger sequencing, and 17 short tandem repeats (STRs). Our approach allowed us to identify five subhaplogroups. Q-M3 and Q-CTS2730/Z780 are undoubtedly autochthonous lineages and represent the most frequent subhaplogroups, with significant representation in self-defined aboriginal populations, and their autochthonous status has been previously described. The aim of present work was to identify the continental origin of the remaining Q lineages. Thus, we analyzed the STR haplotypes for the samples and compared them with haplotypes described by other authors for the rest of the world. Even when haplogroup Q lineages have been extensively studied in America, some of them could have their origin in post-Columbian human migration from Europe and Middle East.

Sex Determination from Fragmented and Degenerated DNA by Amplified Product-Length Polymorphism Bidirectional SNP Analysis of Amelogenin and SRY Genes

Sex determination is important in archeology and anthropology for the study of past societies, cultures, and human activities. Sex determination is also one of the most important components of individual identification in criminal investigations. We developed a new method of sex determination by detecting a single-nucleotide polymorphism in the amelogenin gene using amplified product-length polymorphisms in combination with sex-determining region Y analysis. We particularly focused on the most common types of postmortem DNA damage in ancient and forensic samples: fragmentation and nucleotide modification resulting from deamination. Amplicon size was designed to be less than 60 bp to make the method more useful for analyzing degraded DNA samples. All DNA samples collected from eight Japanese individuals (four male, four female) were evaluated correctly using our method. The detection limit for accurate sex determination was determined to be 20 pg of DNA. We compared our new method with commercial short tandem repeat analysis kits using DNA samples artificially fragmented by ultraviolet irradiation. Our novel method was the most robust for highly fragmented DNA samples. To deal with allelic dropout resulting from deamination, we adopted “bidirectional analysis,” which analyzed samples from both sense and antisense strands. This new method was applied to 14 Jomon individuals (3500-year-old bone samples) whose sex had been identified morphologically. We could correctly identify the sex of 11 out of 14 individuals. These results show that our method is reliable for the sex determination of highly degenerated samples.

Genotyping of the c.1423C>T (p.P475S) polymorphism in the ADAMTS13 gene by APLP and HRM assays: Northeastern Asian origin of the mutant.

ADAMTS13 is a von Willebrand factor-cleaving protease. The mutant types of p.P475S (c.1423C>T) polymorphism in ADAMTS13 have a reduced activity in comparison with the wild type. In the present study, we investigated the frequency of the C-to-T substitution in 2584 genomic DNA samples from 25 Asian, European, and African populations using APLP (amplified product length polymorphism) and/or HRM (high-resolution melting) assays. Allele T (ADAMTS13(∗)T) was detected only in Asian populations and its frequency was observed to decrease gradually from north to south in 24 East Asian populations. Almost all ADAMTS13(∗)T were associated with ABO(∗)O. These results suggested that ADAMTS13(∗)T had occurred on a chromosome with ABO(∗)O in a northern part of East Asia. This SNP is useful as an ancestry-informative marker, and the present genotyping techniques are applicable to the investigation of an association between this SNP and aortic dissection (Kobayashi et al., 2012).

The art of traditional native PAGE: The APLP 48-ID assay for human identification.

When full STR profiles cannot be obtained, further DNA analyses targeting single nucleotide polymorphisms (SNPs) may occasionally yield valuable information. Although the discrimination power of each SNP is relatively low, combined analysis of many SNPs can improve the personal identification ability to a level as high as that of commercial STR typing kits. In this study, we developed a new SNP typing method, named the amplified-product length polymorphism (APLP) 48-ID assay, for genotyping of 47 autosomal SNPs and two X and Y chromosomal markers for sex typing. Forty-seven SNPs were selected from all 22 autosomes, showing high diversity in European, Nigerian, Han Chinese, and Japanese population in the HapMap data. PCR primers were designed to generate amplicons 40-100 bp in length to increase the robustness of the PCR. The APLP 48-ID assay consisted of four independent PCR reactions followed by electrophoretic run on four lanes in a polyacrylamide gel. Complete profiles were obtained when more than 1.2 ng of DNA was used. We applied this assay for genotyping of 236 Japanese individuals. The random matching probability was 3.3E-20, and the power of exclusion was greater than 0.9999999. This method is a rapid, robust, and cost-effective approach for human identification and paternity testing.

The global distribution of the p.R1193Q polymorphism in the SCN5A gene.

The SCN5A (sodium channel, voltage-gated, type V, alpha subunit) gene encodes the cardiac sodium channel, a member of the voltage-gated sodium channel family. The p.R1193Q (c.3578G>A) polymorphism in SCN5A is known to accelerate inactivation of the sodium channel current, and has been identified in patients with Brugada and long QT syndromes. In the present study, we investigated the frequency of the p.R1193Q substitution in more than 4000 genomic DNA samples from 34 Asian, European, and African populations using TaqMan and/or APLP (amplified product length polymorphism) assays. Allele A (p.1193Q) was detected in most Asian populations, but was sporadically observed or absent in European and African populations. These results demonstrated that the p.R1193Q substitution is characteristic of Asian populations.

Mitochondrial DNA haplogrouping of the brown bear,Ursus arctos(Carnivora: Ursidae) in Asia, based on a newly developed APLP analysis

Sequence analyses of the complete brown bear, Ursus arctos, mitochondrial DNA (mtDNA) genome have detected scattered single nucleotide polymorphisms (SNPs) that define distinct mtDNA haplogroups in phylogeographical studies. The degraded DNA in historical samples, such as stuffed or excavated specimens, however, is often not suitable for sequence analyses. To address this problem, we developed an amplified product length polymorphism (APLP) analysis for mtDNA-haplogrouping U. arctos specimens by detecting haplogroup-specific SNPs. We verified the validity and utility of this method by analysing up to 170-year-old skin samples from U. arctos specimens collected widely across continental Eurasia. We detected some of the same haplogroups as those occurring in eastern Hokkaido (Japan) and eastern Alaska in continental Eurasia (the Altai and the Caucasus). Our results show that U. arctos in eastern Hokkaido and eastern Alaska descended from a common ancestor in continental Eurasia, and suggest that U. arctos occupied several refugia in southern Asia during the Last Glacial Maximum. © 2014 The Linnean Society of London, Biological Journal of the Linnean Society, 2014, 111, 627-635.

The Resveratrol Attenuates Ethanol‐Induced Hepatocyte Apoptosis Via Inhibiting ER‐Related Caspase‐12 Activation and PDE Activity In Vitro

Endoplasmic reticulum (ER) stress plays a key role in cell apoptosis pathways. Caspase-12, a proapoptotic gene induced by ER stress, is also the key molecule in ER-related apoptosis. The purpose of this study is to evaluate the protective activity and possible mechanism of resveratrol (ResV) against ethanol (EtOH)-induced apoptosis in human hepatocyte Chang cell line. The human hepatocyte Chang cell line was used to test the hypothesis that ResV may alleviate the liver cell apoptosis induced by EtOH. ER stress-inducible proteins and silent mating type information regulation 2 homolog 1 (SIRT1) were assayed by Western blot. Cell viability was studied by MTT assay and apoptosis was measured by Annexin-V and propidium iodide assay. Caspase-12 activation was examined by immunofluorescence staining. Alcohol dehydrogenase-2 (ADH-2) and aldehyde dehydrogenase-2 (ALDH-2) were measured by polymerase chain reaction amplified product length polymorphism. Phosphodiesterase (PDE) activity was assayed in cell lysates using a cyclic nucleotide PDE assay. EtOH exposure significantly increased the expression of ER stress markers and activated signaling pathways associated with ER stress. These include GRP78, p-IRE1α, p-eIF2α, p-PERK, ATF4 as well as cleaved caspase-3/12, CHOP/GADD153, and Bax in human hepatocyte Chang cell line. The expression of these proteins were significantly down-regulated by ResV (10μM) in a SIRT1-dependent manner. ResV can inhibit EtOH-, tunicamycin-, thapsigargin-induced caspase-12 activation. ADH-2 and ALDH-2 activities are lower in this cell line. PDE activity increased by EtOH was inhibited by ResV (10μM). The results indicate that (i) EtOH-induced activation of caspase-12 could be one of the underlying mechanisms of hepatocyte apoptosis; (ii) EtOH-induced cell apoptosis was alleviated via ResV (10μM) by inhibiting ER stress and caspase-12 activation in a SIRT1-dependent manner; and (iii) SIRT1 activated indirectly by ResV (10μM) attenuates EtOH-induced hepatocyte apoptosis partly through inhibiting PDE activity.

Maternal identification of hybrid eggs in Hexagrammos spp. by means of multiplex amplified product length polymorphism of mitochondrial DNA

Hybridization has been frequently observed among 3 species of greenlings (genus Hexagrammos) common in waters off Japan. In order to estimate the frequency of hybridization events from egg masses collected from male territories, efficient maternal identification of numerous egg masses is required. A novel streamlined approach for maternal identification of 3 Hexagrammos spp. was developed using multiplex amplified product length polymorphism (APLP) analysis of the mitochondrial cytochrome b (Cytb) and the 12S and 16S ribosomal RNA (12-16S rRNA) regions. Concurrent use of species-specific primer sets permits the amplification of different-sized PCR products, diagnosing each species through one procedure of PCR in a single reaction tube. The APLP method produced more rapid, reliable, and cost-efficient species identifications compared to those from an established restriction fragment length polymorphism (RFLP) protocol.

Multiplex amplified product-length polymorphism analysis of 36 mitochondrial single-nucleotide polymorphisms for haplogrouping of East Asian populations

We present a reliable, rapid, and economical multiplex amplified product-length polymorphism (APLP) method for analyzing the haplogroup-diagnostic mitochondrial single-nucleotide polymorphisms (mtSNPs) in East Asian populations. By examining only 36 haplogroup-specific mtSNPs in the coding region by using four 9-multiplex polymerase chain reaction (PCR) and subsequent electrophoresis, we could safely assign 1815 individuals from 8 populations of Japanese, Korean, Chinese, and Germans to 45 relevant haplogroups. This multiplex APLP analysis of coding-region mtSNPs for haplogrouping is especially useful not only for molecular phylogenetic studies but also for large-scale association studies due to its rapid and economical nature. This is the first panel of mtSNPs in the coding region to be used for haplogrouping of East Asian populations.

AB0 Blood Subgroup Allele Frequencies in the Turkish Population

We determined the AB0 blood group system with a PCR based technique termed APLP (Amplified Product Length Polymorphism) in the Turkish population. The method includes ten different allele specific primers and permits identification of the major AB0 genotypes and its suballeles (A1-A2-B-0A-02-0G-AG). The suballeles were amplified in a single tube reaction. We have determined AB0 phenotypes in 129 Turkish individuals. No significant deviation from the Hardy-Weinberg equilibrium was observed.

Multiplex amplified product-length polymorphism analysis for rapid detection of human mitochondrial DNA variations.

A number of mutations in coding and noncoding regions of mitochondrial DNA (mtDNA) have previously been studied. In the present study, we simultaneously typed six mutation sites in the coding region by use of amplified product-length polymorphism (APLP) analysis. The mtDNA variations of 2471 individuals from 20 populations of Japanese, Korean, Chinese, and German were examined and classified into 18 haplotypes. Two of these haplotypes, B1 (estimated ancestral haplotype) and C1, were distributed among all populations tested. However, the haplotypes A1, A2, B2, B3, and C2 were mostly restricted to the Mongoloid populations, whereas haplotypes B5 and C5 appeared almost exclusively in the German population. Phylogenetic analysis by the neighbor-joining method revealed that the Japanese populations were more closely related to each other than to the other East Asian populations surveyed. The multiplex APLP method is suitable for large-scale screening studies of mtDNA variability because it is both rapid and economical.

A case of DNA analysis of ABO blood group variant allele A 2

The ABO phenotype of a bloodstain (B) on a knife that was used as a weapon in an attempted murder case was found to be different from that of the Peruvian victim's blood (AB). Serological analysis showed that the A-antigenicity was much weaker than B antigenicity, suggesting that the victim's phenotype was A 2B or A 3B. So, the ABO genotypes of the knife bloodstain and the victim's blood were determined by DNA analysis. The 261st G deletion, specific to the O 1 allele, was not detected in the specimens by restriction fragment length polymorphism analysis. Also, the 871st A, specific to the A 3 allele, was not found by the allele-specific amplification method. Amplified product length polymorphism and direct sequencing methods finally demonstrated that the typical B sequence was found in one allele and a single C deletion in the 1,059th–1,061st C stretch in the other allele, indicating that the ABO phenotype of the bloodstain and victim's blood were A 2B.

Determination of the HUMTH01 alleles by the APLP method.

We present a simple and rapid technique for determination of alleles at the HUMTH01 locus. The amplified product length polymorphism (APLP) method using two primers different in length, permits the differentiation between allele 9.3 and other alleles. The primers were designed to have an allele-specific nucleotide at the 3' terminal and 11 non-complementary nucleotides were added to the 5' terminal of one of the primers for the allele 9.3. The amplified fragment sizes for the alleles 9.3 and 10 were 80 bp and 70 bp, respectively. This method has proved to be very useful for forensic applications.

Simultaneous Determination of STR Polymorphism and a New Nucleotide Substitution in Its Flanking Region at the CD4 Locus

In the course of the investigation of a pentanucleotide repeat polymorphism at the human CD4 locus, a C-A transversion was found at the position corresponding to the 3' end of the original forward primer presented by Edwards et al. (1). In the present study, the simultaneous determination of the new sequence polymorphism and the pentanucleotide repeat polymorphism at the CD4 locus was attempted. To achieve this purpose, we adopted amplified product length polymorphism (APLP) analysis and designed some new allele-specific forward primers tagged with non-complementary nucleotides differing in length. A total of 646 DNA samples from peripheral blood of Japanese, Chinese and German populations were investigated. Although the C-A transversion was restricted to CD4*5, a new subtype allele with A and 5 repeats, designated CD4*5A, was observed at polymorphic frequencies in the three populations. The simultaneous genotyping by APLP analysis resulted in dramatically increased heterozygosity and discriminating power of the human CD4 locus.

Simultaneous genotyping of alcohol dehydrogenase 2 (ADH2) and aldehyde dehydrogenase 2 (ALDH2) loci by amplified product length polymorphism (APLP) analysis.

Genotyping of the alcohol dehydrogenase 2 (ADH2) and aldehyde dehydrogenase 2 (ALDH2) loci is important in alcohol studies. We describe a method for simultaneous genotyping of ADH2 and ALDH2 based on amplified product length polymorphism (APLP) analysis. Two polymerase chain reaction (PCR) fragments for ADH2 (57 bp, 53 bp) and two for ALDH2 (78 bp, 73 bp) are simultaneously amplified. Nine banding patterns reflecting the genotypes of the ADH2 and ALDH2 loci are clearly and unambiguously distinguished. The APLP method seems to be particularly suited for large-scale population studies of ADH2 and ALDH2 loci because it is simple and rapid.

Rapid communication: a PCR amplified product length polymorphism (APLP) marker at the porcine glucosephosphate isomerase locus shows between-population frequency variation.

Rapid communication: a PCR amplified product length polymorphism (APLP) marker at the porcine glucosephosphate isomerase locus shows between-population frequency variation.

Amplified product length polymorphism (APLP): a novel strategy for genotyping the ABO blood group

We present a simple rapid reproducible polymerase chain reaction based technique, termed amplified product length polymorphism (APLP), as a new strategy for primer design for ABO genotyping. The method involves the use of primers differing in length and permits the identification of the major ABO genotypes (A1, A2, B, OA, OG, and O2) according to the molecular size of the allele-specific amplified products. Ten different primers designed to analyze the variations in nucleotide positions 261, 297, 796, 802, and 1059-1061 of cDNA are mixed in one reaction, and the amplified products are resolved on a polyacrylamide gel. Of eight PCR fragments (132 bp, 120 bp, 108 bp, 98 bp, 88 bp, 80 bp, 72 bp, and 64 bp), two to five are amplified in the reaction according to ABO genotypes. The new technique has been successfully applied to the genotyping of 221 peripheral blood samples from Japanese and Germans whose ABO phenotypes had previously been determined; a novel A allele (AG) was found in Japanese individuals.