Non-invasive genotyping of transgenic animals using fecal DNA

For genotyping of transgenic animals, many IACUC guidelines recommend the use of fecal DNA when possible because this approach is non-invasive. Existing methods for extracting fecal DNA may be costly or involve the use of toxic organic solvents. Furthermore, feces contain an abundance of PCR inhibitors that may hinder DNA amplification when they are co-purified with fecal DNA. Here the authors describe a cost-effective, non-toxic method for genotyping transgenic animals by using the reagent AquaStool to extract fecal DNA and remove PCR inhibitors. Genotyping results obtained from fecal DNA samples extracted using AquaStool were reliably accurate when compared with results obtained from tail DNA samples. Because it is non-invasive, the authors believe that use of this method for genotyping transgenic animals using fecal DNA samples may improve animal welfare.     

Evaluation of a high resolution genotyping method for Chlamydia trachomatis using routine clinical samples

Background

Genital chlamydia infection is the most commonly diagnosed sexually transmitted infection in the UK. C. trachomatis genital infections are usually caused by strains which fall into two pathovars: lymphogranuloma venereum (LGV) and the genitourinary genotypes D–K. Although these genotypes can be discriminated by outer membrane protein gene (ompA) sequencing or multi-locus sequence typing (MLST), neither protocol affords the high-resolution genotyping required for local epidemiology and accurate contact-tracing.

Principal Findings

We evaluated variable number tandem repeat (VNTR) and ompA sequencing (now called multi-locus VNTR analysis and ompA or “MLVA-ompA”) to study local epidemiology in Southampton over a period of six months. One hundred and fifty seven endocervical swabs that tested positive for C. trachomatis from both the Southampton genitourinary medicine (GUM) clinic and local GP surgeries were tested by COBAS Taqman 48 (Roche) PCR for the presence of C. trachomatis. Samples tested as positive by the commercial NAATs test were genotyped, where possible, by a MLVA-ompA sequencing technique. Attempts were made to isolate C. trachomatis from all 157 samples in cell culture, and 68 (43%) were successfully recovered by repeatable passage in culture. Of the 157 samples, 93 (i.e. 59%) were fully genotyped by MLVA-ompA. Only one mixed infection (E & D) in a single sample was confirmed. There were two distinct D genotypes for the ompA gene. Most frequent ompA genotypes were D, E and F, comprising 20%, 41% and 16% of the type-able samples respectively. Within all genotypes we detected numerous MLVA sub-types.

Conclusions

Amongst the common genotypes, there are a significant number of defined MLVA sub-types, which may reflect particular background demographics including age group, geography, high-risk sexual behavior, and sexual networks.     

A simple method for cloning Giardia duodenalis from cultures and fecal samples

Using a novel method for cloning Giardia duodenalis from cultures and fecal samples, 47 clones from 7 isolates were established in vitro. Average colony-forming efficiency in established cultures was 43.2% compared to 11.2% when cloning directly from excystation. The highest success rate of cloning was found with the Portland (P1, ATCC No. 30888) isolate, with a colony-forming efficiency of 92.7%. Cloned and parent populations were compared over a range of 13 enzymes using starch gel electrophoresis. No genetic difference was found between any of the clones and the parent isolates.     

A protocol for isolating putative Helicobacter pylori from fecal specimens and genotyping using vacA alleles

Background. This study outlines steps for isolating and culturing Helicobacter pylori from freshly voided fecal specimens and genotyping isolates for vacA alleles. Materials and methods. A family with four H. pylori‐infected members participated in this pilot study. Criterion for participation was a positive test for H. pylori by the urea breath test. Fecal specimens from children were taken from a freshly soiled diaper, placed in cold buffer, and prepared for culture in less than 2 hours. Culturing of H. pylori utilized selective culture media and isolates were screened for negative Gram stain, positive catalase and oxidase tests, and positive H. pylori 16S ribosomal RNA polymerase chain reaction (PCR). Strain types were determined by vacA genotyping. Results. The isolation procedure is relatively simple, although 5–7 days are required for H. pylori culturing. Isolation and purification of DNA eliminated PCR inhibitors and resulted in reliable analyses. All four family members were infected with the same H. pylori strain with a genotype of vacA s1a/m2. Conclusion. This research lays the foundation for developing a routine and direct noninvasive method to detect the presence of H. pylori in fecal specimens. It is especially convenient for diagnosing children and infants, as samples can be obtained from soiled diapers. Culturing H. pylori from fecal samples in certain cases is important for antibiotic resistant studies prior to treating infected patients and for strain genotyping in epidemiological studies to determine transmission.     

High through-put characterization of insect morphocryptic entities by a non-invasive method using direct-PCR of fecal DNA

The development of non-invasive molecular techniques is currently increasing, particularly in the fields of behavioural ecology and conservation genetics of mammals. Surprisingly, genetic studies of Arthropods and particularly the insects have not benefited yet from the contributions that non-invasive methods have made. Here, we outline a strategy for identifying phytophagous insect genetic entities based on direct-PCR of fecal DNA combined with double strand conformation polymorphism (DSCP) typing. This allows the differentiation of morphocryptic entities within the species Ceutorhynchus assimilis (Coleoptera: Curculionidae), a candidate biocontrol agent of a noxious weed. The results obtained clearly demonstrate the potential for this method to provide a valuable means for genetic and ecological studies of Arthropods.     

Single-nucleotide-polymorphism genotyping for whole-genome-amplified samples using automated fluorescence correlation spectroscopy

Whole-genome amplification (WGA) methods were adopted for single-nucleotide-polymorphism (SNP) typing to minimize the amount of genomic DNA that has to be used in typing for thousands of different SNPs in large-scale studies; 5-10 ng of genomic DNA was amplified by a WGA method (improved primer-extension-preamplification-polymerase chain reaction (I-PEP-PCR), degenerated oligonucleotide primer-PCR (DOP-PCR), or multiple displacement amplification (MDA)). Using 1/100 to 1/500 amounts of the whole-genome-amplified products as templates, subsequent analyses were successfully performed. SNPs were genotyped by the sequence-specific primer (SSP)-PCR method followed by fluorescence correlation spectroscopy (FCS). The typing results were evaluated for four different SNPs on tumor necrosis factor receptor 1 and 2 genes (TNFR1 and TNFR2). The genotypes determined by the SSP-FCS method using the WGA products were 100% in concordance with those determined by nucleotide sequencing using genomic DNAs. We have already carried out typing of more than 300 different SNPs and are currently performing 7,500-10,000 typings per day using WGA samples from patients with several common diseases. WGA coupled with FCS allows specific and high-throughput genotyping of thousands of samples for thousands of different SNPs.     

A rapid, hygienic method for the preparation of fecal samples for liquid scintillation counting.

A method for the preparation of fecal samples for liquid scintillation counting is described which is rapid, hygienic, and inexpensive. By the use of a novel type of homogenizer, fecal samples can be homogenized while totally enclosed within a sealed, plastic bag, so reducing the possible risk of infection. The subsequent preparation of a clear solution suitable for liquid seintillation counting is performed using an “in-vial” digestion technique which enables any ¹⁴CO₂ released during digestion to be trapped within the vial.     

A method for kappa-casein genotyping of bulls

A method for kappa-casein genotyping in bulls has been developed. By analysis of DNA polymorphisms we are able to discriminate between the kappa-casein variant A and B in the bulls. This method will be an efficient tool in selection for the most desirable kappa-casein variant.     

A method for K-casein genotyping of bulls

Summary. A method for K-casein genotyping in bulls has been developed. By analysis of DNA polymorphisms we are able to discriminate between the K-casein variant A and B in the bulls. This method will be an efficient tool in selection for the most desirable K-casein variant.     

A microsampling method for genotyping coral symbionts

Genotypic characterization of Symbiodinium symbionts in hard corals has routinely involved coring, or the removal of branches or a piece of the coral colony. These methods can potentially underestimate the complexity of the Symbiodinium community structure and may produce lesions. This study demonstrates that microscale sampling of individual coral polyps provided sufficient DNA for identifying zooxanthellae clades by RFLP analyses, and subclades through the use of PCR amplification of the ITS-2 region of rDNA and denaturing-gradient gel electrophoresis. Using this technique it was possible to detect distinct ITS-2 types of Symbiodinium from two or three adjacent coral polyps. These methods can be used to intensely sample coral-symbiont population/communities while causing minimal damage. The effectiveness and fine scale capabilities of these methods were demonstrated by sampling and identifying phylotypes of Symbiodinium clades A, B, and C that co-reside within a single Montastraea faveolata colony.     

Morphometric analyses of non-invasive fecal samples of the Korean long-tailed goral (Naemorhedus caudatus) for species and age identification

The Korean long-tailed goral (Naemorhedus caudatus) is at risk of population decline due to habitat loss and fragmentation. Therefore, it is essential to ascertain its presence and/or the identity of individuals of the goral using non-invasive fecal samples for its conservation and management. In this study, we examined the morphology of fecal samples to provide the baseline data that can be used to distinguish species and age of goral individuals. We detected a significant difference in the length-to-width ratios of feces among the five ungulate species found in Korea. Also, we detected a significant difference in the length-to-width ratios of feces of gorals depending on the age groups. To assess the accuracy of species and age identification based on the fecal morphology, we conducted a series of blind comparison between the mean length-to-width ratios of the fecal pellets and the reference mean ratio values of the fecal pellets. Using 20 fecal pellets, our results showed 73%–86% probability of correct identification of the three species (gorals, goats, and roe deer), and 83%–90% probability of correct identification of >5 year-old goral individuals. The use of fecal morphometric analyses will be useful for the studies of Korean ungulate species, particularly the endangered gorals.     

SNP genotyping for the genetic monitoring of laboratory mice by using a microarray-based method with dualcolour fluorescence hybridisation

Ensuring the genetic homogeneity of the mice used in laboratory experiments contributes to the Reduction aspect of the Three Rs, by maximising the quality of the data obtained from any animals that are used for these purposes, and ultimately reducing the numbers of animals used. Single nucleotide polymorphism (SNP) genotyping is especially suitable for use in the analysis of the genetic purity of model organisms such as the mouse, because bi-allelic markers remain fully informative when used to characterise crosses between inbred strains. Here, we attempted to apply a microarray-based method for a SNP marker to monitor the genetic quality of inbred mouse strains, so as to validate the reliability, stability and applicability of this SNP genotyping panel. The amplified PCR products containing four different SNP loci from four inbred mouse strains were spotted and immobilised onto amino-modified glass slides to generate a microarray. This was then interrogated through hybridisation with dual-colour probes, to determine the SNP genotypes of each sample. The results indicated that this microarray-based method could effectively determine the genotypes of the four selected SNPs with a high degree of accuracy. We have developed a new SNP genotyping technique for effective use in the genetic monitoring of inbred mouse strains.     

PCR method for genotyping and zygosity-testing of RasH2 transgenic mice.

In short-term carcinogenicity testing using CB6F1-TgrasH2 mice, sibling nonTgrasH2 mice are used as a negative control. However, selection of TgrasH2 and nonTgrasH2 mice has been performed by PCR with only transgene specific primers by the conventional method. Therefore, the conventional method involves the risk of false negative results due to reaction failure, and contamination with TgrasH2 mice in the control mice group. Based on the nucleotide sequence information around the pre-integration site, we developed a genotyping method for distinguishing not only TgrasH2 mice (hemizygous for the Tg allele) but also nonTgrasH2 (homozygous for the nonTg allele) in a positive manner.     

Sex identification of wolf (Canis lupus) using non-invasive samples

We have developed new specific primers for sex determination from forensic samples of wolves (Canis lupus), such as hair, saliva, faecal, tooth and urine samples. In order to improve molecular sexing, we performed a multiplex semi-nested polymerase chain reaction (PCR) and several replicated amplifications per sample to avoid errors in low quantity DNA samples, such as allelic dropout and false alleles. The sex of individuals is automatically determined by capillary electrophoresis with a fluorescently labelled internal sex-specific primer from each pair. Our method yielded sex identification on 100% of invasive samples and 93% of forensic samples, being one of the highest success rates obtained from wild animals.     

A non-invasive method for measuring preimplantation embryo physiology

The physiology of the early embryo may be indicative of embryo vitality and therefore methods for non-invasively monitoring physiological parameters from embryos could improve preimplantation diagnoses. The self-referencing electrophysiological technique is capable of non-invasive measurement of the physiology of individual cells by monitoring the movement of ions and molecules between the cell and the surrounding media. Here we use this technique to monitor gradients of calcium, potassium, oxygen and hydrogen peroxide around individual mouse preimplantation embryos. The calcium-sensitive electrode in self-referencing mode identified a region of elevated calcium concentration (approximately 0.25 pmol) surrounding each embryo. The calcium gradient surrounding embryos was relatively steep, such that the region of elevated calcium extended into the medium only 4 microns from the embryo. By contrast, using an oxygen-sensitive electrode an extensive gradient of reduced dissolved oxygen concentration was measured surrounding the embryo and extended tens of micrometres into the medium. A gradient of neither potassium nor hydrogen peroxide was observed around unperturbed embryos. We also demonstrate that monitoring the physiology of embryos using the self-referencing technique does not compromise their subsequent development. Blastocyts studied with the self-referencing technique implanted and developed to term at the same frequency as did unexamined, control embryos. Therefore, the self-referencing electrode provides a valuable non-invasive technique for studying the physiology and pathophysiology of individual embryos without hindering their subsequent development.     

A multiple-tubes approach for accurate genotyping of very small DNA samples by using PCR: statistical considerations.

A multiple-tubes procedure is described for using PCR to determine the genotype of a very small DNA sample. The procedure involves dividing the sample among several tubes, then amplifying and typing the contents of each tube separately. The results are analyzed by a statistical procedure which determines whether a genotype can be conclusively assigned to the DNA sample. Simulation studies show that this procedure usually gives correct results even when the number of double-stranded fragments in the sample is as small as 30. The procedure remains effective even in the presence of small amounts of laboratory contamination. We find that the multiple-tubes procedure is superior to the standard one-tube procedure, either when the sample is small or when laboratory contamination is a potential problem; and we recommend its use in these situations. Because the procedure is statistical, it allows the degree of certainty in the result to be quantified and may be useful in other PCR applications as well.     

A single nucleotide polymorphism genotyping method using phosphate-affinity polyacrylamide gel electrophoresis

To date, various methods have been developed to facilitate the genotyping of a single nucleotide polymorphism (SNP) for aiding in the diagnosis and treatment of inherited diseases. The most commonly used method for SNP genotyping is an allele-specific hybridization procedure using an expensive fluorochrome-labeled oligonucleotide probe and a specialized fluorescence analyzer. Here, we introduce a simple and reliable genotyping method using a 1:1 mixture of 5'-phosphate-labeled and nonlabeled allele-specific polymerase chain reaction (PCR) primers. The method is based on the difference in mobility of the phosphorylated and nonphosphorylated PCR products (in the same number of basepairs) on phosphate-affinity polyacrylamide gel electrophoresis. The phosphate-affinity site is a polyacrylamide-bound dinuclear zinc(II) complex, which preferentially captures the 5'-phosphate-labeled allele-specific product compared with the corresponding nonlabeled product. The obtained DNA migration bands can be visualized by ethidium bromide staining. We demonstrate the genotyping of a SNP reported in a human cardiac sodium channel gene, SCN5A, using this novel procedure.