Charlotte Hallenberg

Multiplex PCR and minisequencing of SNPs— a model with 35 Y chromosome SNPs

We have developed a robust single nucleotide polymorphism (SNPs) typing assay with co-amplification of 25 DNA-fragments and the detection of 35 human Y chromosome SNPs. The sizes of the PCR products ranged from 79 to 186 base pairs. PCR primers were designed to have a theoretical Tm of 60 +/- 5 degrees C at a salt concentration of 180 mM. The sizes of the primers ranged from 19 to 34 nucleotides. The concentration of amplification primers was adjusted to obtain balanced amounts of PCR products in 8mM MgCl2. For routine purposes, 1 ng of genomic DNA was amplified and the lower limit was approximately 100 pg DNA. The minisequencing reactions were performed simultaneously for all 35 SNPs with fluorescently labelled dideoxynucleotides. The size of the minisequencing primers ranged from 19 to 106 nucleotides. The minisequencing reactions were analysed by capillary electrophoresis and multicolour fluorescence detection. Female DNA did not influence the results of Y chromosome SNP typing when added in concentrations more than 300 times the concentrations of male DNA. The frequencies of the 35 SNPs were determined in 194 male Danes. The gene diversity of the SNPs ranged from 0.01 to 0.5.

Paternity Testing Commission of the International Society of Forensic Genetics: recommendations on genetic investigations in paternity cases.

The International Society for Forensic Genetics (ISFG) has established a Paternity Testing Commission (PTC) with the purpose of formulating international recommendations concerning genetic investigations in paternity testing. The PTC recommends that paternity testing be performed in accordance with the ISO 17025 standards. The ISO 17025 standards are general standards for testing laboratories and the PTC offers explanations and recommendations concerning selected areas of special importance to paternity testing.

High frequencies of Y chromosome lineages characterized by E3b1, DYS19-11, DYS392-12 in Somali males

We genotyped 45 biallelic markers and 11 STR systems on the Y chromosome in 201 male Somalis. In addition, 65 sub-Saharan Western Africans, 59 Turks and 64 Iraqis were typed for the biallelic Y chromosome markers. In Somalis, 14 Y chromosome haplogroups were identified including E3b1 (77.6%) and K2 (10.4%). The haplogroup E3b1 with the rare DYS19-11 allele (also called the E3b1 cluster γ) was found in 75.1% of male Somalis, and 70.6% of Somali Y chromosomes were E3b1, DYS19-11, DYS392-12, DYS437-14, DYS438-11 and DYS393-13. The haplotype diversity of eight Y-STRs (‘minimal haplotype’) was 0.9575 compared to an average of 0.9974 and 0.9996 in European and Asian populations. In sub-Saharan Western Africans, only four haplogroups were identified. The West African clade E3a was found in 89.2% of the samples and the haplogroup E3b1 was not observed. In Turks, 12 haplogroups were found including J2*(xJ2f2) (27.1%), R1b3*(xR1b3d, R1b3f) (20.3%), E3b3 and R1a1*(xR1a1b) (both 11.9%). In Iraqis, 12 haplogroups were identified including J2*(xJ2f2) (29.7%) and J*(xJ2) (26.6%). The data suggest that the male Somali population is a branch of the East African population – closely related to the Oromos in Ethiopia and North Kenya – with predominant E3b1 cluster γ lineages that were introduced into the Somali population 4000–5000 years ago, and that the Somali male population has approximately 15% Y chromosomes from Eurasia and approximately 5% from sub-Saharan Africa.

A report of the 2002–2008 paternity testing workshops of the English speaking working group of the International Society for Forensic Genetics

The English Speaking Working Group (ESWG) of the International Society for Forensic Genetics (ISFG) offers an annual Paternity Testing Workshop open to all members of the group. Blood samples, a questionnaire and a paper challenge are sent to the participants. Here, we present the results of the 2002-2008 Paternity Testing Workshops with the objective to evaluate the uniformity of DNA-profiling and conclusions of the participating laboratories as well as to clarify tendencies in typing strategies and biostatistical evaluations of the laboratories. The numbers of participating laboratories increased from 46 in 2002 to 68 in 2008. The results showed an increasing degree of concordance concerning methods and DNA systems used and a high degree of uniformity in typing results with discrepancies in 0.1 and 0.3 % of all submitted PCR-based results. The paper challenges showed uniformity in the calculation of the weight of evidence for simple cases with straight-forward genetic constellations. However, a high degree of variation existed in complex scenarios with rare genetic constellations such as genetic inconsistencies/possible silent alleles, rare alleles and haplotypes.

Additional assignment of the human 5S rRNA genes to chromosome region 1q31

A major fraction of the human 5S rRNA genes has previously been assigned to chromosome region 1q42.11-->q42.13 (Sørensen et al., 1991). Through in situ hybridization of different tritiated probes to metaphase spreads, we have demonstrated that a minor fraction of the 5S rRNA genes is localized at band 1q31. This fraction amounts to 25-30% of the genes found in the region 1q42.11-->q42.13. Results obtained with the chromosomes of a balanced translocation carrier involving this region indicate that the major cluster is localized in band 1q42.13.

Porcine 5S rRNA genes map to 14q23 revealing syntenic relation to human HSPA6- and 7

5S rRNA is one of the two small RNA molecules localized in the large ribosomal subunit and thereby is involved in protein synthesis. 5S rRNAs from many different organisms have been sequenced, and more than 700 5S rRNA transcripts and 5S rRNA gene sequences have been compiled (Specht et al. 1991). The 5S rRNA genes have been studied in a large number of organisms (reviewed in Korn 1982; Geiduscheck and Tocchini-Valentini 1988; Willis 1993). Compared with the lower eukaryotes, thorough knowledge of the 5S rRNA genes in mammals is scarce. The

A report of the 2000 and 2001 paternity testing workshops of the English speaking working group of the international society for forensic genetics.

During the last 10 years, the English Speaking Working Group (ESWG) of the International Society for Forensic Genetics (ISFG) has once a year arranged a Paternity Testing Workshop in which blood samples as well as a questionnaire concerning laboratory strategies were distributed to the participating laboratories. In 2000 and 2001, paper challenges were included in the workshops. Here, we present the results of the 2000 and 2001 Paternity Testing Workshops. The numbers of participating laboratories were 33 (2000) and 36 (2001). A total of 36% (2000) and 31% (2001) of the laboratories submitted typing results of variable number of tandem repeats (VNTRs) investigated with restriction fragment length polymorphism (RFLP) and single locus probes (SLPs). A total of 91% (2000) and 86% (2001) submitted typing results of polymerase chain reaction (PCR) based systems. Typing errors occurred in 0.3% of the submitted PCR-based results in 2000 and in 0.1% in 2001. The results of the paper challenges showed a high degree of variation in the formulas used for calculation of the weight of evidence of rare events such as inconsistencies or possible silent alleles. The majority of the laboratories used the same formulas for calculations of frequently occurring events.

Effect of mutations in the upstream promoter on the transcription of human 5S rRNA genes

The human 5S rRNA gene has a 12-mer external promoter, the D box, localized about 30 bp upstream the coding sequence. By site directed mutagenesis 58 different D box promoter mutants were made. While some mutations in the D box allowed full transcription, other mutations decreased the transcriptional activity to 20-50% compared to the bona fide gene, showing the importance of this external promoter in transcription initiation. A number of maxi 5S rRNA genes were constructed from bona fide genes and D box mutated clones. Transfection of HeLa cells with maxi 5S rRNA genes showed that the D box is also important for 5S rRNA gene expression in vivo. Evidence from different eukaryotic cells suggests that expression of 5S rRNA genes is regulated by external promoters in addition to the internal control region.

Concordance study and population frequencies for 16 autosomal STRs analyzed with PowerPlex® ESI 17 and AmpFℓSTR® NGM SElect™ in Somalis, Danes and Greenlanders

A concordance study of the results of PowerPlex(®) ESI 17 and AmpFℓSTR(®) NGM SElect™ kits obtained from 591 individuals from Somalia (N=198), Denmark (N=199) and Greenland (N=194) was performed. Among 9456 STR types, seven discordant results were found with the two kits: one observed in the D19S433 system in an individual from Denmark and six in the SE33 system in six individuals from Somalia. Sequencing of SE33 in the six samples with discordant results showed G>A transition 15bp downstream of the repeat unit in three of the individuals, and G>A transition 68bp downstream of the repeat unit in the other three individuals. Population data for 16 autosomal STR systems analyzed in 989 individuals from Somalia, Denmark and Greenland are also presented. The highest mean heterozygosity was observed in Danes (82.5%). With the exception of D8S1179 in Danes, no significant deviations from Hardy-Weinberg expectations were observed. Only one pair of systems (D12S391 and D18S51) showed significant allelic association in Greenlanders (after Holm-Šidák correction). A MDS plot drawn from pairwise FST values calculated between 21 populations showed a clear displacement of the Greenlandic population versus the other ones included in the analyses. The highest combined chance of exclusion and power of discrimination was observed for Danes reaching values of 99.9999987% and 1 in 1.8×10(21), respectively.

Y-chromosome STR haplotypes in males from Greenland.

A total of 272 males from Greenland were typed for 11 Y-chromosome STRs DYS19, DYS385a/b, DYS389-I, DYS389-II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439 with the PowerPlex Y System (Promega). A total of 146 different haplotypes were observed and the haplotype diversity was 0.9887. The number of haplotypes seen once was 108 and the most common haplotype was observed in 12 males. A significant F(ST) value was observed (F(ST)=0.012, P<0.00001) when comparing the population of 15 locations in Greenland assigned to 7 groups. The significance could mainly be attributed to the subpopulation of males from Tasiilaq (East of Greenland). The R(ST) value was not statistically significant (R(ST)=0.016, P=0.15).