RESUMEN
In criminal investigations there are some cases in which identifying the presence of vaginal secretions provides crucial evidence in proving sexual assault. However, there are no methods for definitively identifying vaginal secretions. In the present study, we focused on Lactobacillus levels in vaginal secretions and developed a novel identification method for vaginal secretions by relative quantification based on real time PCR. We designed a Lactobacillus conserved region primer pair (LCP) by aligning 16S rRNA gene sequences from major vaginal Lactobacillus species (Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners and Lactobacillus jensenii), and selected the human specific primer pair (HSP) as an endogenous control for relative quantification. As a result, the ΔCt (ΔCt=Ct[LCP]-Ct[HSP]) values of vaginal secretions (11 out of 12 samples) were significantly lower than those of saliva, semen and skin surface samples, and it was possible to discriminate between vaginal secretions and other body fluids. For the one remaining sample, it was confirmed that the predominant species in the microflora was not of the Lactobacillus genus. The ΔCt values in this study were calculated when the total DNA input used from the vaginal secretions was 10pg or more. Additionally, the ΔCt values of samples up to 6-months-old, which were kept at room temperature, remained unchanged. Thus, we concluded in this study that the simple ΔCt method by real time PCR is a useful tool for detecting the presence of vaginal secretions.
Asunto(s)
ADN Bacteriano/análisis , Lactobacillus/genética , Vagina/metabolismo , Secuencia de Bases , Cartilla de ADN , ADN Bacteriano/genética , Femenino , Humanos , ARN Ribosómico 16S/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Vagina/microbiologíaRESUMEN
One of the alleles which leads to ninth component of complement deficiency (C9D) is R95X (nt343C>T), which is present in most cases of C9D in Japan. In this study, we carried out nt343C>T typing by the method of polymerase chain reaction with sequence-specific primers (PCR-SSP), and showed the frequency of the R95X allele in German, Italian, Thai, Korean and Chinese populations. We did not find the R95X allele in the German or Italian populations. The allele frequency of R95X in the three Asian populations is as follows: Thais 0.019, Koreans 0.008, and Chinese 0.002. As the allele frequency in the Japanese population is 0.036, the results provide supporting evidence that the R95X is an allele characteristic of Japanese.
Asunto(s)
Dermatoglifia del ADN , Etnicidad/genética , Frecuencia de los Genes , Alelos , Pueblo Asiatico/genética , Complemento C9/deficiencia , Complemento C9/genética , Cartilla de ADN , Homocigoto , Humanos , Reacción en Cadena de la Polimerasa , Población Blanca/genéticaRESUMEN
We previously described two haplotypes named the ABORR*L-associated and ABORR*S-associated haplotypes in the 5'-upstream region of the ABO blood group gene. Here we studied polymorphisms in exons (Exs) 3 and 4 and introns (Ints) 2 and 3 of the ABO gene, and analyzed the haplotypes in those Exs, Ints, and the 5'-upstream region. Two haplotypes (at Int2nt108-Int2nt362-Int2nt369-Int2nt539-Ex3nt106-Int3nt1178-Int3nt1357-Ex4nt188-Ex4nt189) were deduced to be (1) A-C-C-C-T-C-T-A-T, which was linked with ABORR*L and ABO*O(A), and (2) A-C-C-C-G-T-C-G-C, G-C-C-C-G-T-C-G-C, and A-T-G-A-G-T-C-G-C, which were linked with ABORR*S and the other common ABO alleles. This finding also shows the existence of two major lineages of the Japanese ABO alleles.
Asunto(s)
Sistema del Grupo Sanguíneo ABO/genética , Haplotipos , Alelos , Pueblo Asiatico/genética , Exones , Humanos , Intrones , Japón , Reacción en Cadena de la Polimerasa , Polimorfismo GenéticoRESUMEN
We performed genotyping of the ABO system in Italian and Israeli population samples. The nucleotides at 11 positions, nts 261, 297, 467, 526, 646, 681, 703, 796, 802, 803 and 1060, were analyzed by PCR-RFLP, PCR-SSP and PCR direct sequencing methods. We found three rare ABO alleles besides the common alleles (*)A1(Pro) (=(*)A101), (*)A2(Leu) (=(*)A201), (*)B (=(*)B101), (*)O(T) (=(*)O01), (*)O(A) (=(*)O02) and (*)O2 (=(*)O03), but did not detect ( *)A1(Leu) (=( *)A102) which is a common allele in Asians. The rare alleles were tentatively named (*)Ov1, (*)Ov2, and (*)Bv. As ( *)Bv has been found in two Japanese individuals and (*)O2 is not a rare ABO allele in Europeans, not only (*)O2 but also the (*)Ov1 and (*)Ov2 alleles may be characteristic of European populations.
Asunto(s)
Sistema del Grupo Sanguíneo ABO/genética , Alelos , Población Blanca/genética , Frecuencia de los Genes , Genotipo , Humanos , Israel , Italia , Reacción en Cadena de la Polimerasa , Análisis de SecuenciaRESUMEN
To examine the X-inactivation patterns of normal human nails, we performed the human androgen receptor gene assay of DNA samples extracted separately from each finger and toe nail plates of nine female volunteers. The X-inactivation pattern of each nail was unique and constant for at least 2 years. The frequency of nails with one of the two X-chromosomes exclusively inactivated was 25.9%. In the nails composed of two types of cells with either one X-chromosome inactivated, the two cell types were distributed in patchy mosaics. These findings suggest that the composition of precursor cells of each nail is maintained at each site at least through several cycles of regeneration time, and that the nail plate has a longitudinal band pattern, each band consisting of cells with only one of the two X-chromosomes inactivated. Using the frequency of nails with one of two X-chromosomes exclusively inactivated, we estimated the number of progenitor cells that gave rise to the nail plate during development to be about 3, under the assumption that the process follows the binominal distribution model. A strong correlation observed among the big, index and little fingers, and among the corresponding toes suggests an interesting interpretation concerning their morphogenetic process.
Asunto(s)
Uñas/metabolismo , Inactivación del Cromosoma X , Femenino , Humanos , Masculino , Uñas/citología , Uñas/embriología , Receptores Androgénicos/genética , Células MadreRESUMEN
We investigated the polymorphisms in the 5'-upstream region between nucleotide position (nt) -9600 and nt-4105 of the ABO blood group gene using PCR and direct sequencing methods. We found 16 single nucleotide polymorphisms, two insertion-deletion polymorphisms and two sequence polymorphisms. One of the insertion-deletion polymorphisms was found at nts from -9605 to -9204, and the alleles of that locus were named ABORR*L (non-deletion) and ABORR*S (52-base-deletion). There were two haplotypes constructed from 14 polymorphisms in the region between nt-9600 and nt-7565; they were tentatively named ABORR*L-associated and ABORR*S-associated haplotypes. The ABORR*L-associated haplotype may link with ABO*O(A) (also known as ABO*O201), and the ABORR*S-associated haplotype links with the other common alleles. This indicates the existence of two major lineages of the Japanese ABO alleles in the 5'-upstream region from nt-7565. In contrast to these findings, we observed six haplotypes in the region between nt-6371 and nt-4105, and we assume that the sequence in that region is variable as compared with those in the other 5'-upstream regions. We examined the generation of two sequence polymorphisms found in the present study. In the both cases, the formation of a hairpin loop caused by palindrome in a single-stranded DNA molecule may play an important role in generating the polymorphism.
Asunto(s)
Región de Flanqueo 5'/genética , Sistema del Grupo Sanguíneo ABO/genética , Haplotipos , Polimorfismo de Nucleótido Simple , Pueblo Asiatico/genética , Humanos , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
We analyzed the single nucleotide polymorphisms (SNPs) in the sixth (C6) and the seventh (C7) component genes of the complement system in a sample of the Japanese population, using polymerase chain reaction (PCR)-based methods and PCR direct sequencing. SNPs in the C6 gene studied here are as follows: A413C in exon 3, T1674C in exon 10, T7145A in exon 13, G[357+32]A in intron 2, and G[503-78]A in intron 3. We confirmed that nt413A and nt413C were associated with C6A and C6B, respectively. The result of the nt2145 typing showed that two subtypes exist in the C6B allotype. The SNP of G[357+32]A in intron 2 could be analyzed by using the PCR-RFLP method with HinfI. Allele frequencies in the Japanese population were found to be *G=0.920 and *A=0.080. SNPs in the C7 gene are as follows: T382C in exon 4, G1166C and A1258C in exon 9, and G[+10]A in intron 13. Nt382C and nt1258C would be responsible for C7-5 (=C7-3) and C7-4 allotypes, respectively.
Asunto(s)
Complemento C6/genética , Complemento C7/genética , Exones , Genética de Población , Polimorfismo de Nucleótido Simple , Dermatoglifia del ADN/métodos , Frecuencia de los Genes , Humanos , Japón , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADNRESUMEN
Exon 1 of the androgen receptor (AR) gene on the X chromosome contains a polymorphic CAG trinucleotide short tandem repeat. We describe here the rapid and reliable method of typing CAG repeats using electrophoresis, with denaturing polyacrylamide gel and an allelic ladder marker. Twenty-one alleles (the repeat number ranges from 13 to 35) were found using CAG repeat typing in normal Japanese individuals (83 males and 82 females). The allelic diversity (h) calculated was h=0.889, illustrating that CAG repeats at the AR locus is a highly polymorphic system.
Asunto(s)
Exones , Receptores Androgénicos/genética , Secuencias Repetidas en Tándem , Cromosomas Humanos X , Electroforesis en Gel de Poliacrilamida , Femenino , Frecuencia de los Genes , Humanos , Masculino , Reacción en Cadena de la Polimerasa , Polimorfismo Genético , Análisis de Secuencia de ADNRESUMEN
The ABO blood group system is important in forensic genetics, as well as transfusion medicine. Since the elucidation of the molecular basis of ABO gene regulation, nucleotides of variant alleles or suballeles have been analyzed by polymerase chain reaction (PCR)-based methods and sequencing. Ael (A-elute) is one of the subgroups of A in the ABO system. By analyzing the suballeles responsible for Ael phenotype by PCR-RFLP and PCR direct sequencing, we found seven types of Ael allele. The allele frequency of ABO*Ael in a Japanese population was calculated to be 0.0049.
Asunto(s)
Sistema del Grupo Sanguíneo ABO/genética , Alelos , Etnicidad/genética , Polimorfismo de Longitud del Fragmento de Restricción , Humanos , Japón , Fenotipo , Reacción en Cadena de la Polimerasa/métodosRESUMEN
Complement C7 is one of the components of membrane attack complex (MAC) generated by the terminal complement cascade. C7 protein is polymorphic and most of its polymorphisms have been identified using isoelectric focusing (IEF), which detects protein charge differences. To date, the molecular bases of the polymorphisms detected by IEF have not been determined. In this paper, we describe the structural bases of two C7 IEF-detected polymorphisms, C7*3 and C7*4, both of which are common in Asian populations. C7*3 resulted from substitution of cysteine (Cys) at amino acid residue 106 by charged arginine (Arg; C106R), while charged lysine (Lys) at amino acid residue 398 was replaced by neutral glutamine (Gln; K398Q) in C7*4. As C7*3 is hypomorphic, it is important to study its possible associations with diseases such as immunological disorders and infections. We present genetic bases for this C7 polymorphism, which we determined using polymerase chain reaction (PCR)-based genotyping, a simple and accurate method suitable for large-scale studies.