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The aim of this experiment was to measure enteric methane (CH4) emission and its relation with rumen digestion in dairy cows fed diets rich in 1 of the 2 carbohydrate sources, starch or sugar. The rations were based on late first-cut grass–clover silage supplemented with wheat (Wh), NaOH-treated wheat (Wh+NaOH), sugar beet molasses (Mo), or sugar beet molasses with addition of sodium bicarbonate (Mo+Bic). Wheat and molasses made up 35% of dry matter in the 2 diets with molasses and wheat, respectively. Four cows fitted with ruminal, duodenal, and ileal canulae were used in a 4 × 4 Latin square design. Nutrient digestibility was measured using chromium oxide and titanium oxide as flow markers, and emissions of CH4 and hydrogen were measured via open-circuit indirect calorimetry on 4 consecutive days. Data were analyzed using PROC MIXED of SAS (version 9.4; SAS Institute Inc., Cary, NC) with treatment and period as fixed effects and cow as random effect. Furthermore, orthogonal contrasts were calculated. The cows produced 32.5, 33.6, 36.2, and 35.1 L of CH4/kg of dry matter intake (DMI) on diets Wh, Wh+NaOH, Mo, and Mo+Bic, respectively. The emission of CH4 per day, per kilogram of DMI, and per kilogram of energy-corrected milk as well as daily hydrogen emission were higher on the Mo diet compared with the Wh diet. With the present inclusion of wheat and molasses in the diet, no effects of NaOH treatment of wheat or of sodium bicarbonate supplementation to the Mo diet could be demonstrated on CH4 emission expressed per kilogram of DMI or per kilogram of energy-corrected milk. The duodenal flow of starch was higher when wheat was treated with NaOH. Under the conditions in the present experiment, ruminal NDF digestibility was not affected by carbohydrate source, NaOH treatment of wheat, or bicarbonate supplementation. Total volatile fatty acid concentration in the rumen and the proportions of acetate and propionate were not affected by carbohydrate source, NaOH treatment of wheat, or bicarbonate supplementation. Likewise, we could not show any influence of diet on microbial protein synthesis or efficiency of microbial protein synthesis expressed as grams of microbial protein synthesis per kilogram of true rumen-digested organic matter. We concluded that CH4 emission was increased when wheat was replaced by molasses, whereas no effect of manipulating rumen fermentation by NaOH treatment of wheat or addition of bicarbonate to molasses could be found with a level of approximately 25% of dry matter from starch and sugar, respectively. [ABSTRACT FROM AUTHOR]

This review explores the key factors that influence the optimization, routine use, and profile interpretation of the SNaPshot single-base extension (SBE) system applied to forensic single-nucleotide polymorphism (SNP) genotyping. Despite being a mainly complimentary DNA genotyping technique to routine STR profiling, use of SNaPshot is an important part of the development of SNP sets for a wide range of forensic applications with these markers, from genotyping highly degraded DNA with very short amplicons to the introduction of SNPs to ascertain the ancestry and physical characteristics of an unidentified contact trace donor. However, this technology, as resourceful as it is, displays several features that depart from the usual STR genotyping far enough to demand a certain degree of expertise from the forensic analyst before tackling the complex casework on which SNaPshot application provides an advantage. In order to provide the basis for developing such expertise, we cover in this paper the most challenging aspects of the SNaPshot technology, focusing on the steps taken to design primer sets, optimize the PCR and single-base extension chemistries, and the important features of the peak patterns observed in typical forensic SNP profiles using SNaPshot. With that purpose in mind, we provide guidelines and troubleshooting for multiplex-SNaPshot–oriented primer design and the resulting capillary electrophoresis (CE) profile interpretation (covering the most commonly observed artifacts and expected departures from the ideal conditions). [ABSTRACT FROM AUTHOR]

Abstract: Fundamental to most forensic analyses is the availability of genomic DNA of adequate quality and quantity. To perform a multitude of genetic analyses and assays requires a sufficiently large amount of template. However, DNA yield from forensic samples is frequently limiting the extent of genetic typing. A possible solution to overcome this “bottleneck” of forensic and paleoarcheological DNA analyses could be the amplification of the entire genomic DNA prior to locus specific PCR analysis. Whole Genome Amplification appears to be a promising tool to obtain sufficient DNA amounts from forensic samples of limited quantity. [Copyright &y& Elsevier]

Abstract: We performed multiple displacement amplification (MDA) of 50 blood and 50 saliva samples placed on FTA cards. A 1.2-mm disk was punched out of the FTA cards. The disk was washed, dried and used as target for the GenomiPhi DNA amplification kit. On average, the Phi29 polymerase produced 2 μg whole genome amplified DNA (wgaDNA) with DNA fragment sizes ranging from a few hundred bp to 12 kbp. A total of 1–2 ng wgaDNA was typed using the AmpFlSTR® SGM Plus™ amplification kit and the resulting STR profiles analysed and compared to genotypes obtained from genomic DNA. Only 25 of the 100 wgaDNA samples gave concordant STR profiles. Locus drop-outs, missing alleles, allele imbalances or false alleles were observed in 11, 27, 61 and 10 wgaDNA samples, respectively. The same FTA disk was used successfully in five consecutive rounds of MDA. However, after the 4th and 5th round of MDA, a further increase in the number of allele drop-outs as well as system and allele imbalances were observed. [Copyright &y& Elsevier]

Abstract: Fifty Danish paternity cases from the year 2004 were selected based on the results obtained with the AmpFlSTR® Identifiler® PCR amplification kit. In all cases, the calculated paternity index (PI) was higher than 10,000, and there was not observed any genetic inconsistency between mother and child, or between father and child. DNA from the selected trios was used to type 7 VNTRs (D2S44, D5S43, D5S110, D7S21, D7S22, D12S11 and D16S309) with the RFLP technique and 52 SNPs using a PCR multiplex with 52 PCR primer pairs and two SBE multiplexes with 23 and 29 SBE primers, respectively. PIs were calculated based on each set of loci (STRs, VNTRs and SNPs). On average, the typical PI obtained from analyses of 15 STRs was 4 times higher than the typical PI obtained from analyses of 7 VNTRs, and 17 times higher than the typical PI obtained from analyses of 52 SNPs. However, in only 3 trios analyzed for SNPs and 1 trio analyzed for VNTRs, PI<10,000. No genetic inconsistency was detected between mother and child or between father and child in the SNP-analyses. In contrast, one father–child and one mother–child genetic inconsistency were observed in the VNTR-analyses. [Copyright &y& Elsevier]

We have developed a PCR-based assay with co-amplification of 25 DNA fragments and detection of 35 Y chromosome SNPs with the SNaPshot technique. The Y SNP package can define 34 Y chromosome haplogroups and it can identify the majority of the Y chromosome haplogroups of interest in the populations relevant to forensic genetics in Denmark. We typed 194 Danes, 215 Greenlanders, and 201 Somalis, all males. A total of 21 different haplogroups were identified. In Danes, 11 haplogroups with frequencies from 0.5% to 38% were identified and three of the haplogroups, I, R1b*(xR1b1, R1b6, R1b8) and R1a1*(xR1a1b), were found in ∼91% of the population. In Greenlanders, 10 haplogroups with frequencies from 0.5% to 50% were identified and the haplogroups P*(xQ3a, R1), R1b*(xR1b1, R1b6, R1b8) and I, were found in ∼86% of the population. In Somalis, 14 haplogroups with frequencies from 0.5% to 79% were identified and the haplogroups E3b1*(xE3b1b) and K*(xN3, O, P) were found in ∼88% of the population. The distribution of haplogroups was compared to the distribution found in 65 males from West Africa. [Copyright &y& Elsevier]

Despite ample focus on this endangered species, conservation planning for chimpanzees residing outside Africa has proven a challenge because of the lack of ancestry information. Here, we analysed the largest number of chimpanzee samples to date, examining microsatellites in >100 chimpanzees from the range of the species in Africa, and 20% of the European zoo population. We applied the knowledge about subspecies differentiation throughout equatorial Africa to assign origin to chimpanzees in the largest conservation management programme globally. A total of 63% of the genotyped chimpanzees from the European zoos could be assigned to one of the recognized subspecies. The majority being of West African origin (40%) will help consolidate the current breeding programme for this subspecies and the identification of individuals belonging to the two other subspecies so far found in European zoos can form the basis for breeding programmes for these. Individuals of various degree of mixed ancestry made up 37% of the genotyped European zoo population and thus highlight the need for appropriate management programmes guided by genetic analysis to preserve maximum genetic diversity and reduce hybridization among subspecies. [ABSTRACT FROM AUTHOR]

A novel single-base extension (SBE) assay using cleavable and noncleavable SBE primers in the same reaction mix is described. The cleavable SBE primers consisted of deoxyribonucleotides and one ribonucleotide (hereafter denoted chimeric primers), whereas the noncleavable SBE primers consisted of only deoxyribonucleotides (hereafter denoted standard primers). Biotin-labeled dd-NTPs were used in the SBE reaction, and the SBE products were purified using the monomeric avidin triethylamine purification protocol, ensuring that only primers extended with a biotin-ddNTP in the 3'-end were isolated. A ribonuclease mix was developed to specifically cleave the chimeric primers, irrespective of the base of the ribonucleotide, whereas standard primers without a ribonucleotide were unaffected by the ribonuclease treatment. The SBE products were analyzed in linear mode using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. The cleaved SBE products were detected in the 2000-5500 m/z range, and the noncleaved SBE products were detected in the 5500-10000 m/z range. The method was validated by typing 17Y chromosome single-nucleotide polymorphisms in 100 males with a 17-plex SBE package containing 9 chimeric primers and 8 standard primers. [ABSTRACT FROM AUTHOR]

One of the most promising techniques for typing of multiple single-nucleofide polymorphism (SNP) is detection of single base extension primers (SBE) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We present a new MALDITOF MS protocol for typing of multiple SNPs in a single reaction. Biotin-labeled ddNTPs were used in the SBE reaction and solid phase-bound monomeric avidin was used as capturing/purification scheme allowing the exclusive release of the SBE products under gentle condidons using 5% triethylantine. We dubbed this method monomeric avidin triethylamine purification. The biotinlabeled dd.NTPs contained linkers with different masses ensuring a clear separation of the alleles even for SBE primers with a mass of 10 300 Da. Furthermore, only 25-350 fmol of SBE primers were necessary in order to obtain reproducible MALDI-TOF spectra. Similar signal intensities were obtained in the 5500-10 300 m/z mass range by increasing the concentration of the longer SBE primers in the reaction. To validate the technique, 17 Y-chromosome SNPs were analyzed in 200 males. The precision and accuracy of the mass determination were analyzed by parametric statistic, and the potential use of MALDI-TOF MS for SNP typing is discussed. [ABSTRACT FROM AUTHOR]

The objectives of the present study were: (i) to evaluate a novel procedure for catheterization of the mammary artery; (ii) to measure amino acid, glucose and calcium arteriovenous differences in lactating sows; and (iii) to evaluate the use of different markers in estimating mammary plasma flow by the Fick principle. Five multiparous sows were fitted with two catheters, in the cranial epigastric artery and anterior mammary vein. Catheterization of the cranial epigastric artery was carried out with a new surgical technique. Implanted catheters remained functional until day 15 (in two animals), day 21 (one animal) and day 27 (one animal), whereas in one case this technique failed. Sampling of arterial and venous blood took place on days 9, 15, 21 and 27 of lactation. Mean extraction rates±SEM (percent) were 31.2±3.1 arginine (Arg), 16.4±2.5 glutamine (Gln), 27.2±5.7 threonine (Thr), 8.3±2.6 glycine (Gly), 12.4±2.7 alanine (Ala), 20.2±3.4 tyrosine (Tyr), 18.1±2.8 proline (Pro), 28.2±6.2 methionine (Met), 18.5±2.2 valine (Val), 26.4±3.1 phenylalanine (Phe), 30.2±3.1 isoleucine (Ile), 41.4±3.4 leucine (Leu), 32.2±4.0 lysine (Lys) and 20.0±2.76 glucose. There was a relatively high abundance of lysine in milk compared with the amount extracted from plasma. This suggests lysine to be limiting in the present experimental situation, which was probably due to a low feed intake only providing 31.4 g Lys day[sup -1] on average. Mean mammary plasma flow (litres plasma day[sup -1]), evaluated by use of Lys, Met and calcium as markers in the metabolic Fick principle, amounted to 7869±989 Lys, 6391±2139 Met and 4059±747 calcium. The large difference in the results implies that there is a need for other markers to measure the mammary plasma flow or novel methods based on other principles to measure mammary plasma flow, which is an important factor in quantitative studies of daily mammary uptake of nutrients. [ABSTRACT FROM AUTHOR]

Introduction of new methods requires meticulous evaluation before they can be applied to forensic genetic case work. Here, a custom QIAseq Targeted DNA panel with 164 ancestry informative markers was assessed using the MiSeq sequencing platform. Concordance, sensitivity, and the capability for analysis of mixtures were tested. The assay gave reproducible and nearly concordant results with an input of 10 and 2 ng DNA. Lower DNA input led to an increase in both locus and allele drop-outs, and a higher variation in heterozygote balance. Locus or allele drop-outs in the samples with less than 2 ng DNA input were not necessarily associated with the overall performance of a locus. Thus, the QIAseq assay will be difficult to implement in a forensic genetic setting where the sample material is often scarce and of poor quality. With equal or near equal mixture ratios, the mixture DNA profiles were easily identified by an increased number of imbalanced heterozygotes. For more skewed mixture ratios, the mixture DNA profiles were identified by an increased noise level. Lastly, individuals from Great Britain and the Middle East were investigated. The Middle Eastern individuals showed a greater affinity with South European populations compared to North European populations. [ABSTRACT FROM AUTHOR]

The EUROFORGEN NAME panel is a regional ancestry panel designed to differentiate individuals from the Middle East, North Africa, and Europe. The first version of the panel was developed for the MassARRAY system and included 111 SNPs. Here, a custom AmpliSeq EUROFORGEN NAME panel with 102 of the original 111 loci was used to sequence 1098 individuals from 14 populations from Europe, the Middle East, North Africa, North-East Africa, and South-Central Asia. These samples were also sequenced with a global ancestry panel, the Precision ID Ancestry Panel. The GenoGeographer software was used to assign the AIM profiles to reference populations and calculate the weight of the evidence as likelihood ratios. The combination of the EUROFORGEN NAME and Precision ID Ancestry panels led to fewer ambiguous assignments, especially for individuals from the Middle East and South-Central Asia. The likelihood ratios showed that North African individuals could be separated from European and Middle Eastern individuals using the Precision ID Ancestry Panel. The separation improved with the addition of the EUROFORGEN NAME panel. The analyses also showed that the separation of Middle Eastern populations from European and South-Central Asian populations was challenging even when both panels were applied. [ABSTRACT FROM AUTHOR]

Abstract: In this study, the GenomiPhi™ DNA Amplification Kit (Amersham Biosciences) was used to investigate the potential of whole genome amplification (WGA) when considering samples originating from more than one donor. DNA was extracted from blood samples, quantified and normalised before being mixed in ratios of 1:1, 1:3, 1:7 and 1:15. Proportions were checked using standard STR analysis before being subjected to WGA. Once amplified using GenomiPhi™, the ratios were again checked. Relative proportions were found to be maintained in the 1:1 and 1:3 ratios following WGA; the observed peak ratios were found to match the expected peak ratios regardless of the starting concentration of DNA. With samples mixed in the ratio of 1:7 and 1:15, and when the concentration of starting material was at the manufacturer''s lower limit, too few minor component peaks were found to allow for statistical analysis. With an initial template exceeding 1 ng/μL there was an increase in problems associated with profile interpretation but the results obtained indicated that mixture proportions could be quantifiably maintained. [Copyright &y& Elsevier]

Abstract: We have implemented a laboratory automation system (LAS) for the preparation of database samples. The LAS encompass registration of samples in a LIMS database, export of a work-list to a puncher, punching of FTA-cards by a BSD600-duet puncher, electronic sample check by means of barcodes, washing of punches by the THEONYX liquid-handler from MWG, PCR amplification and electrophoresis of amplicons. We validated the system using fresh blood and saliva immobilized on FTA-cards as sources of biological material. The use of the LAS led to: [–] Correct STR types with a quality equal to or higher than that of the profiles produced using chelex-extracted DNA. [–] A reduction of approximately 35% in the number of reanalysed samples. [–] A reduced risk of mix-up of samples during the laboratory procedure. [–] Fewer sample transfers. [–] A reduction in the number of PCR cycles from 28 to 24 compared to chelex-extracted DNA from blood. During the validation process, we did not observe mix-up of samples, loss of FTA-card punches, contamination from external sources, or cross-contamination between samples. [Copyright &y& Elsevier]

Abstract: The Melanocortin 1 Receptor (MC1R) is an important regulator of pigmentation in hair and skin. MC1R mutations D84C, R151C, R160W, D294H have been designated strong red hair alleles (R) and V60L, V92M, R163Q as weak red hair alleles (r). We have developed a package of 15 SNPs from the MC1R gene and typed 34 Danes with red hair and 56 Danes with blond and dark hair, 65 of the individuals were related in 8 families. In our model, we designated R142H, 29InsA and 179InsC as R alleles. Only red-haired individuals were homozygous for R or compound heterozygous for two R alleles, and the wt/wt genotype was observed only in samples from individuals with blond or dark hair. [Copyright &y& Elsevier]

Abstract: An efficient method that can be used to simultaneously amplify a set of genetic loci across the genome with high reliability can provide a valuable tool for single nucleotide polymorphism (SNP) forensic genotyping. A crucial element is the number of individual biochemical reactions that must be performed. The SNPforID consortium (www.snpforid.org) was established in 2003 with the principal goal of developing a SNP-based system of DNA analysis that would have comparable discrimination power and ease of use to those of existing short tandem repeat (STR) based techniques. Here, we describe a strategy for amplifying 52 genomic DNA fragments, each containing one SNP, in a single tube, and accurately genotyping the PCR product mixture using two single base extension reactions. This multiplex approach reduces the cost of SNP genotyping and requires as little as 0.5 ng of genomic DNA to detect 52 SNPs. We used a multiple injection approach for DNA sequencers that can effectively detect all the SNPs amplified in a single electrophoretic run. We present SNP data for 700 unrelated individuals from 9 populations. [Copyright &y& Elsevier]

Single nucleotide polymorphism (SNP) typing was done with a PCR-based assay with co-amplification of 25 DNA fragments and detection of 35 Y chromosome SNPs by means of minisequencing with Snapshot technique. The Y SNP package can identify 34 Y chromosome haplogroups including the majority of the Y chromosome haplogroups of interest in the Canary Islands population. A total of 348 and 269 unrelated Canary Islands males were typed for 35 Y-SNPs and 8 Y-STRs, respectively. We observed 16 Y chromosome SNP haplogroups with frequencies from 0.3% to 48%. The majority of the individuals carried the Y chromosome haplogroup R1b or I of European origin while ∼13% and ∼7% of the Y chromosome haplogroups found indicated African and Middle Eastern heritage. Further diversity was detected when additional typing of Y chromosome STR markers was performed. [Copyright &y& Elsevier]

Matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry (MS) offers a reliable, inexpensive and time-saving method for SNP genotyping. The method is based on weight determination of single base extended (SBE) primers. Here, we present a MALDI-TOF protocol for typing 17 Y chromosome SNPs using biotin-labelled ddNTP in the SBE reaction and solid-phase avidin capture purification of extended primers. All 17 fragments were amplified in a multiplex reaction and all 17 primers were extended in a single SBE reaction. [Copyright &y& Elsevier]

Some STR loci have internal sequence variations, which are not revealed by the standard STR typing methods used in forensic genetics (PCR and fragment length analysis by capillary electrophoresis (CE)). Typing of STRs with next-generation sequencing (NGS) uncovers the sequence variation in the repeat region and in the flanking regions. In this study, 363 Danish individuals were typed for 56 STRs (26 autosomal STRs, 24 Y-STRs, and 6 X-STRs) using the ForenSeq™ DNA Signature Prep Kit to establish a Danish STR sequence database. Increased allelic diversity was observed in 34 STRs by the PCR-NGS assay. The largest increases were found in DYS389II and D12S391, where the numbers of sequenced alleles were around four times larger than the numbers of alleles determined by repeat length alone. Thirteen SNPs and one InDel were identified in the flanking regions of 12 STRs. Furthermore, 36 single positions and five longer stretches in the STR flanking regions were found to have dubious genotyping quality. The combined match probability of the 26 autosomal STRs was 10,000 times larger using the PCR-NGS assay than by using PCR-CE. The typical paternity indices for trios and duos were 500 and 100 times larger, respectively, than those obtained with PCR-CE. The assay also amplified 94 SNPs selected for human identification. Eleven of these loci were not in Hardy-Weinberg equilibrium in the Danish population, most likely because the minimum threshold for allele calling (30 reads) in the ForenSeq™ Universal Analysis Software was too low and frequent allele dropouts were not detected. [ABSTRACT FROM AUTHOR]

BACKGROUND: The 2 most common hemoglobinopathies, sickle cell trait and alpha (+)-thalassemia, confer partial resistance to fatal forms of malaria, but the molecular basis for this protection is still not understood. Examination of the relationship between these traits and malaria transmission intensity may provide insights into the protection afforded. METHODS: The distribution of the 2 traits was assessed among children resident in 13 villages in the Eastern Arc Mountains in Tanzania, where Plasmodium falciparum transmission intensity is closely correlated with altitude. Associations between the prevalence of the 2 traits and malariometric indices were investigated by logistic regression. Short tandem repeat (STR) microsatellite allele frequencies were used to assess population substructuring. RESULTS: The frequency of alpha (+)-thalassemia ranged from 10%-25% in high-altitude villages (>1200 m) to 45%-55% in low-altitude villages (<600 m). The carriage rate of alpha (+)-thalassemia decreased by approximately 12% per 100-m increase in altitude (P<.001) and was approximately 50% lower among those with patent parasitemia than among uninfected individuals (P=.014). The prevalence of the sickle cell trait was lower than that of alpha (+)-thalassemia (range, 0%-14%) and was significantly associated with village altitude only (P=.011). STR allele frequencies were similar in all villages. CONCLUSIONS: In this malaria-endemic region of Tanzania, alpha (+)-thalassemia is common and clearly associated with P. falciparum transmission intensity. There was no evidence of population substructuring, and the results are suggestive of selection of the alpha (3.7) allele by malaria. Copyright © 2007 Infectious Diseases Society of America [ABSTRACT FROM AUTHOR]

Abstract: The European Consortium “High throughput analysis of single nucleotide polymorphisms for the forensic identification of persons—SNPforID” has performed a selection of candidate Y-chromosome SNPs (single nucleotide polymorphisms) for making inferences on the geographic origin of an unknown sample. A “Major Y chromosome haplogroup typing kit” has been developed, which allows the multiplex amplification of 29 SNPs in a single reaction followed by a single base extension (SBE) reaction (minisequencing) and separation of the resulting extension products by capillary electrophoresis. [Copyright &y& Elsevier]