Effects of electron-beam irradiation on buccal-cell DNA

Buccal cells were collected from 29 participants, by use of mouthwash rinses, and were split into equal aliquots, with one aliquot irradiated by electron-beam (E-beam) irradiation equivalent to the sterilizing dosage used by the U.S. Postal Service and the other left untreated. Aliquots were extracted and tested for DNA yields (e.g., TaqMan assay for quantifying human genomic DNA), genomic integrity, and amplification-based analysis of genetic variants (e.g., single-nucleotide polymorphisms [SNPs] and single tandem repeats [STRs]). Irradiated aliquots had lower median DNA yields (3.7 microg/aliquot) than untreated aliquots (7.6 microg/aliquot) (P<.0005) and were more likely to have smaller maximum DNA fragment size, on the basis of genomic integrity gels, than untreated aliquots (P<.0005). Irradiated aliquots showed poorer PCR amplification of a 989-bp beta-globin target (97% for weak amplification and 3% for no amplification) than untreated aliquots (7% for weak amplification and 0% for no amplification) (P<.0005), but 536-bp and 268-bp beta-globin targets were amplified from all aliquots. There was no detectable irradiation effect on SNP assays, but there was a significant trend for decreased detection of longer STRs (P=.01) in irradiated versus untreated aliquots. We conclude that E-beam irradiation reduced the yield and quality of buccal-cell specimens, and, although irradiated buccal-cell specimens may retain sufficient DNA integrity for some amplified analyses of many common genomic targets, assays that target longer DNA fragments (>989 bp) or require whole-genome amplification may be compromised.     

DNA extraction from bronchial aspirates for molecular cytology: which method to take?

OBJECTIVE: To date, there are only few systematic reports on the quality of DNA extracted from routine diagnostic cytologic specimens. It was the aim of the present study to evaluate the ability of 50% ethanol/2% carbowax (Saccomanno fixative) to preserve bronchial secretions with high quality genomic DNA as well as to compare different DNA extraction methods. METHODS: DNA was extracted from 45 bronchial aspirates by four different extraction protocols. Beside DNA yield, DNA quality with regard to purity, integrity, and PCR success rate were investigated. RESULTS: No fragmentation of sample DNA due to the fixative was detected. It was preserved as high molecular weight DNA. DNA yield, purity, and integrity were dependent on the DNA extraction method to some extend. Irrespective of the DNA extraction method the PCR success rate for amplification of beta-globin gene fragments (268, 536, and 989 bp) was 100%. CONCLUSION: A fixative containing 50% ethanol/2% carbowax preserves high quality DNA which is well suited for PCR-based assays regardless of the extraction protocol used. The selection of the DNA extraction protocol has to be adjusted to the circumstances of application.     

Prenatal and post-natal screening of β-thalassemia and hemoglobin E genes in Thailand using denaturing high performance liquid chromatography

We have developed methods based on PCR and denaturing high performance liquid chromatography (DHPLC) for rapid identifications of common β-thalassemia mutations found in Thailand. The β-globin gene was separately amplified by PCR on four different fragments covering eight most common β-thalassemia mutations including nucleotide ?28 A-G, codon 17 (A-T), IVSI-1 (G-T), IVSI-5 (G-C), codon 26 (G-A or Hb E), codons 41/42 (–TTCT), codons 71/72 (+A) and IVSII-654 (C-T). After PCR amplification, heteroduplex was generated by denaturation at 95 °C for 5 min followed by a slow reduction in temperature to 25 °C at 0.03 °C/s. Analysis of heteroduplex was done on an automated WAVE Nucleic Acid Fragment Analysis System. Specific DHPLC profile for each mutation was demonstrated which could be used in screening for all eight β-thalassemia mutations. Further validation was done on 42 pre- and post-natal DNA samples which demonstrated 100 % accuracy as compared to the result obtained with conventional PCR assays. In a remaining case with an unknown mutation, a different DHPLC profile was noted on one of the amplified fragment. Further DNA sequencing of this fragment revealed a T-G transversion at the IVSI-116, a previously un-described mutation in Thai population. The DHPLC assay developed should prove useful for rapid screening of known and unknown β-thalassemia mutations during carrier screening and pre-natal diagnosis which would facilitate an ongoing prevention and control program of thalassemia.     

Development of microdissection and chromosome specific genomic library in Lilium tigrinum

Techniques for microdissection and microcloning were established using chromosome 1 of triploid Lilium tigrinum. Chromosome 1 was dissected from a membrane slide using a microbeam system. Digestion with proteinase K was done before PCR amplification for more than 24 hours. The dissected chromosomes were then amplified by degenerate oligonucleotide primed PCR (DOP-PCR) and linker adaptor-mediated PCR (LA-PCR). Successful PCR amplification relied on critical concentrations of both MgCl₂ and Taq polymerase. The optimum concentration of MgCl₂ and Taq polymerase was 2.5 mM and 0.5 U, respectively. Amplification of the dissected chromosome using DOP-PCR had more sensitivity dependent upon PCR factors, but LA-PCR was more dependent on the linker ligation. Amplified DNA products ranged from 100 to 2500 bp both for DOP- and LA-PCR. Evaluated clones only ranged from 100 to 1700 bp for DOP-PCR and 100 to 900 bp for LA-PCR. Based on the sequence results, most of the sequences from the DOP-PCR and LA-PCR showed no significant similarity with known data in NCBI database. However, about 2% of the sequence data was partially matched with plant microsatellites with low similarity. The results derived from the microdissection of a large genome organism such as Lilium showed informative and useful for the development of microsatellite repeat markers. Sequence data from the chromosome specific DNA library was considered for the development of microsatellite markers.     

Improved noninvasive genotyping method: application to brown bear (Ursus arctos) faeces

We redesigned new microsatellite primers and one sex‐specific primer for amplification of faecal DNA from brown bears (Ursus arctos). We also combined a semi‐nested polymerase chain reaction (PCR) with a newly developed multiplex preamplification method in order to increase the quality of the amplified DNA fragments. In comparison with a conventional PCR approach, the genotyping error rate was substantially reduced and the amplification rate was increased. This new approach could be transposed to other species where conventional PCR methods experience low success due to limited DNA concentration and/or quality.     

An optimisation approach to increase DNA amplification success of otter faeces

Faeces have proved to be a suitable non-invasive DNA source for microsatellite analysis in wildlife research. For the success of such studies it is essential to obtain the highest possible PCR amplification success rate. These rates are still relatively low in most carnivorous species, especially in the otter (Lutra lutra). We therefore optimised the entire microsatellite genotyping process by combining our findings with results from previous studies to gain a high rate of reliable genotypes. We investigated the influence of otter faecal quality in relation to the quantity of slimy secretions and three levels of storage periods at ?20°C on amplification success. Further, we tested the cost-effective and time-saving Chelex extraction method against the profitable QIAamp^® DNA Stool Kit (Qiagen), and compared three PCR methods - a standard single-step PCR protocol, a single-locus two-step PCR procedure and a multiplex two-step PCR procedure - regarding success rate and genotyping errors. The highest amplification success rate (median: 94%; mean: 78%) was achieved using faecal samples consisting only of jelly extracted with the QIAamp^® DNA Stool Mini Kit (Qiagen) immediately after collection and amplified following the time and cost efficient multiplex two-step PCR protocol. The two-step procedure, also referred to as pre-amplification approach, turned out to be the main improvement as it increases amplification success about 11% and reduces genotyping errors about 53%, most notably allelic dropouts.     

Identification of novel breast tumor-specific mutation(s) in the q11.2 region of chromosome 17 by RAPD/AP-PCR fingerprinting

Analysis of genetic instability in breast cancer tissues compared to uninvolved breast tissues from the same individuals by RAPD (random amplified polymorphic DNA)/AP-PCR (arbitrarily primed PCR) fingerprinting using 30 arbitrary primers revealed 190 amplified DNA fragments. Presumably, each of these represents a gene locus in a different region of the genome of breast cancer tissues. Among these amplified DNA fragments, 65 (34.2%) exhibited presence and absence or reductions and enhancements in the intensity in breast cancer tissues compared to uninvolved breast tissues from the same individuals, and 11 amplified DNA fragments (5.7%) represented polymorphisms in the uninvolved human breast tissues. Reductions and enhancements in the intensity of some of the amplified fragments were observed indicating allelic gains or losses in the breast tumor genome compared to the matched uninvolved tissue genome. The presence or absence of some of the amplified DNA fragments were observed in this study indicating homozygous deletions or insertions in the breast tumor DNA compared to the matched uninvolved tissue DNA. Notably, an insertion of a 1270 bp amplified fragment was observed in 81% (17 of 21) of the tumor samples using the primer, OPC04. This amplified fragment resolved into two, 1200 and 1300 bp, single-stranded amplified fragments on the denaturing sequencing gel. This separation into single-stranded fragments suggests that the amplified fragment contains a conformation that is semistable. The 1270 bp amplified fragment localizes to the q11.2 region of chromosome 17. Sequence analysis of this fragment showed a significant DNA base sequence similarity (93%) with one of the breast tumor-specific human EST. The similarity with EST sequences and RT-PCR analysis showed that a part of this amplified fragment is from the coding region of the genome. Any one of the events observed in this study could play an important role in the development of breast cancer or could occur during the clonal expansion of the genetically unstable breast cells.     

Identification of genetic markers for Korean native cattle (Hanwoo) by RAPD analysis

In order to develop the specific genetic marker for Korean native cattle (Hanwoo), randomly amplified polymorphic DNA (RAPD) analysis of 6 different cattle breeds was attempted by using 38 decamer primers. In comparison of RAPD patterns, two distinctive DNA bands specific for Hanwoo were detected. One was 296 bp of DNA fragment found to be specific only for female Hanwoo when primer GTCCACACGG was employed. In individual analysis of this RAPD marker was observed only in female individuals with the possibility of 85.3%. The other was 521 bp of RAPD marker amplified using TCGGCGATAG and AGCCAGCGAA primers, which showed 83.0% of genetic frequency in 85 male and 68 female individuals tested. Nucleotide sequencing of these genetic markers revealed that 296 bp marker has a short microsatellite-like sequence, ACCACCACAC, and a tandem repeat sequence of microsatellite GAAAAATG in the determined sequence. Two distinctive tandem repeats of microsatellite sequences, AAC and GAAGA, were also appeared in 521 bp DNA marker. In BLAST search, any gene having high homology with these markers was not found     

不同保存方法对川金丝猴粪便DNA提取效果的影响

目的:川金丝猴(Rhinopithecus roxellana)是我国特有珍稀物种,其粪便作为一种非损伤性样品,为珍稀濒危动物的种群数量调查、遗传多样性评价、亲缘关系、系统进化等研究带来了很大便利,本研究试图建立高效、简便的粪便样品保存方法。方法:在现有珍稀濒危动物粪便样品保存方法的基础上,分别使用干燥法、冷冻法和干燥-冷冻法保存川金丝猴的粪便样品,比较了不同保存方法的DNA提取效果,以及对mtDNA控制区片段的PCR扩增成功率和微卫星基因的PCR扩增效率。结果:干燥法、冷冻法和干燥-冷冻法三种不同保存方法保存粪便1周时间后,提取的粪便DNA样本扩增mtDNA片段的成功率均为92%,微卫星基因的扩增成功率分别为79%、78%、80%;保存2个月后,mtDNA片段扩增成功率分别为80%、76%和80%,微卫星基因扩增成功率分别为65%、61%、67%;保存6个月后,mtDNA片段扩增成功率分别为56%、52%和64%,微卫星基因扩增成功率分别40%、34%、46%。因此,随着保存时间的增长,三种方法的保存效率都将明显降低,但干燥-冷冻法得到的DNA样本扩增成功率相对较高。结论:粪便样品能够为川金丝猴的遗传多样性评价等相关研究提供有效信息,干燥-冷冻法保存能够更为有效的保证DNA的提取和基因扩增效率。     

番木瓜单染色体的显微分离与克隆

用玻璃针显微分离出番木瓜(CaricapapayaL.)单染色体,经过LA-PCR扩增得到80-700bp的DNA片段。Southern杂交表明,扩增片段与番木瓜基因组DNA之间有同源性,从而证明番木瓜单染色体DNA已经被成功扩增。将扩增产物克隆到pGEM-T-Easy载体中,约获得1.18×105个克隆,酶切鉴定插入片段大小为100-400bp。     

An Improved Field Method to Obtain DNA for Individual Identification From Wolf Scat

ABSTRACT Sampling of feces for genetic studies of wild populations can be problematic because of the low quality and quantity of template DNA obtained. We used cotton swabs in the field to isolate the mucous layer on the surface of fresh wolf (Canis lupus, C. lycaon, and their hybrids) scats followed by immediate preservation, and compared microsatellite genotyping of DNA from these fresh field swabs (FS) to that of previously frozen laboratory swabs (LS). In single polymerase chain reactions (PCRs) of 2 multiplexes, amplification at 8 loci was higher in the FS samples (FS = 50%, LS = 15%; P = 0.02) because proportion, quantity, and quality of large fragment wolf nuclear DNA from these samples was greater (2.5–25%, 6.25–62.5 ng/swab, 35% amplified at 1,000 base pairs [bp]) than from the LS samples (1.9%–10%, 4.7–25 ng/swab, 10% amplified at 1,000 bp). Paired blood and fresh field-swabbed samples had identical genotypes. In 84 multiplex PCRs we found no evidence of allelic dropout associated with low template quality or quantity. We conclude that field swabbing of fresh wolf scat facilitates field storage and reduces the need for multiple amplifications at single microsatellite loci, thereby reducing the genotyping costs for wildlife projects that use noninvasive samples.     

Patterns of nuclear DNA degeneration over time--a case study in historic teeth samples

The amount of nuclear DNA extracted from teeth of 279 individual red fox Vulpes vulpes collected over a period spanning the last three decades was determined by quantitative polymerase chain reaction (PCR). Although teeth were autoclaved during initial collection, 73.8% of extracts contained sufficient DNA concentration (> 5 pg/ micro L) suitable for reliable microsatellite genotyping but the quantity of nuclear DNA decayed significantly over time in a nonlinear pattern. The success of PCR amplification across four examined canine microsatellites over time was dependent on fragment size. By including data from two different tests for human contamination and from frequencies of allelic dropout and false alleles, the methodological constraints of population genetic studies using microsatellite loci amplified from historic DNA are discussed.     

The effect of sample age and storage method on DNA yield and microsatellite amplification from baboon (Papio ursinus) faecal samples

Sampling methods that allow DNA collection without the physical handling of animals are popular in conservation genetics, but DNA isolated from faecal samples may be degraded, potentially leading to erroneous microsatellite genotyping results. We collected baboon faecal samples from fresh to 1-week post-defecation in a controlled sampling environment and preserved these using three storage techniques. After DNA isolation and quantification, the samples were genotyped at eight microsatellite loci. Quantitatively, DNA yield was highest when using silica as storage medium. However, microsatellite amplification from samples stored in 95% ETOH were most successful, with 100% success for fresh samples and dropping only marginally to 87.5% of loci for samples collected after 1 week. This disparity between quantitative and qualitative results suggests that total DNA concentrations do not necessarily provide a reliable indication of the amount of target DNA present in a DNA isolate. Our results nevertheless confirm that microsatellite fragments can successfully be amplified from faecal samples up to 1 week post-defecation, with careful selection of storage protocols and loci.     

Extracting DNA from museum bird eggs, and whole genome amplification of archive DNA

We present a comprehensive protocol for extracting DNA from egg membranes and other internal debris recovered from the interior of blown museum bird eggs. A variety of commercially available DNA extraction methods were found to be applicable. DNA sequencing of polymerase chain reaction (PCR) products for a 176‐bp fragment of mitochondrial DNA was successful for most egg samples (> 78%) even though the amount of DNA extracted (mean = 14.71 ± 4.55 ng/µL) was significantly less than that obtained for bird skin samples (mean = 67.88 ± 4.77 ng/µL). For PCR and sequencing of snipe (Gallinago) DNA, we provide eight new primers for the ‘DNA barcode’ region of COI mtDNA. In various combinations, the primers target a range of PCR products sized from 72 bp to the full ‘barcode’ of 751 bp. Not all possible combinations were tested with archive snipe DNA, but we found a significantly better success rate of PCR amplification for a shorter 176‐bp target compared with a larger 288‐bp fragment (67% vs. 39%). Finally, we explored the feasibility of whole genome amplification (WGA) for extending the use of archive DNA in PCR and sequencing applications. Of two WGA approaches, a PCR‐based method was found to be able to amplify whole genomic DNA from archive skins and eggs from museum bird collections. After WGA, significantly more archive egg samples produced visible PCR products on agarose (56.9% before WGA vs. 79.0% after WGA). However, overall sequencing success did not improve significantly (78.8% compared with 83.0%).     

Deficiency of uridine monophosphate synthase (DUMPS) and X-chromosome deletion in fetal mummification in cattle

Ten mummified fetuses were tested for the deficiency of uridine monophosphate synthase (DUMPS), which is known to contribute to the embryonic and fetal mortality in cattle. Genomic DNAs of the mummified fetuses were extracted from tissue samples collected from the mummies and were amplified by GenomiPhi DNA amplification kit. UMPS gene of the mummies was amplified by polymerase chain reaction (PCR) with DUMPS primers. Out of ten mummies examined, two fetuses were heterozygous (carriers) for DUMPS as indicated by the presences of three bands of 89, 53 and 36 bp. Estimated stage of gestation when the death occurred in the two mummies was 3.5 and 2.5 months, respectively. The other fetuses exhibited only two bands of 53 and 36 bp on the polyacrylamide gel indicated that they were normal. On the other hand, all the mummies were sexed using AMX/Y primers. Specific regions of Y and X chromosomes were amplified by PCR using AMX/Y. The expected 280 bp fragment in the female sample and the 280 and 217 bp in the male sample were observed. Nine mummies had a normal X and Y chromosome bands; however, the other mummified fetus exhibited only Y chromosome band, while the constitutive X chromosome fragment was missing. The estimated stage of gestation when the death occurred in this mummified fetus was 100 days. This might be the first report of DUMPS and X-chromosome deletion at the amelogenin gene in bovine-mummified fetuses in Japan.     

How to Open the Treasure Chest? Optimising DNA Extraction from Herbarium Specimens

Herbarium collections are potentially an enormous resource for DNA studies, but the use of herbarium specimens in molecular studies has thus far been slowed down by difficulty in obtaining amplifiable DNA. Here we compare a set of commercially available DNA extraction protocols and their performance in terms of DNA purity and yield, and PCR amplification success as measured by using three differentially sized markers, the rbcL barcoding marker (cpDNA), the LEAFY exon 3 (nrDNA), and the trnL((UAA)) P6 loop (cpDNA). Results reveal large differences between extraction methods, where DNA purity rather than yield is shown to be strongly correlated with PCR success. Amplicon size shows similarly strong correlation with PCR success, with the shortest fragment showing the highest success rate (78%, P6 loop, 10-143 base pairs (bp)) and the largest fragment the lowest success (10%, rbcL, 670 bp). The effect of specimen preparation method on PCR success was also tested. Results show that drying method strongly affects PCR success, especially the availability of fragments longer than 250 bp, where longer fragments are more available for PCR amplification in air dried material compared to alcohol dried specimens. Results from our study indicate that projects relying on poor-quality starting material such as herbarium or scat samples should focus on extracting pure DNA and aim to amplify short target regions (<200-300 bp) in order to maximise outcomes. Development of shorter barcoding regions, or mini-barcodes within existing ones should be of high importance as only a few options are currently available; this is particularly important if we hope to incorporate the millions of herbarium samples available into barcoding initiatives and other molecular studies.     

Preliminary development of a diagnostic test for Brucella using polymerase chain reaction

A highly sensitive and specific diagnostic test for Brucella based on polymerase chain reaction is under development in our laboratory. A commercially available PCR kit was used to create primers that allowed the amplification of a 635 bp fragment of a 43 kDa outer membrane protein gene from Brucella abortus strain 19. We successfully amplified the cloned gene present in the pMS64 plasmid and genomic Brucella S19 DNA. The amplified DNA was easily detected by agarose gel electrophoresis. Using both the pMS64 plasmid and Br. abortus S19 purified DNA as template each component of the PCR reaction was adjusted for the optimum amplification of the DNA sequence. Optimum specific amplification resulted when the primer annealing temperature was 60C. The gene fragment was amplifiable in 25 different Brucella species and strains. To test the specificity of the reaction, DNA extracted from 17 micro-organisms possibly associated with cattle were tested. No amplification was observed. The sensitivity of the reaction was determined with different concentrations of genomic Brucella strain 19 DNA. As little as 0.1 pg DNA (less than 100 brucella cells) could be detected. The specificity and sensitivity of PCR combined with its simplicity and speed suggests the potential of this technique for routine diagnosis of brucellosis.     

Preliminary development of a diagnostic test for Brucella using polymerase chain reaction

A highly sensitive and specific diagnostic test for Brucella based on polymerase chain reaction is under development in our laboratory. A commercially available PCR kit was used to create primers that allowed the amplification of a 635 bp fragment of a 43 kDa outer membrane protein gene from Brucella abortus strain 19. We successfully amplified the cloned gene present in the pMS64 plasmid and genomic Brucella S19 DNA. The amplified DNA was easily detected by agarose gel electrophoresis. Using both the pMS64 plasmid and Br. abortus S19 purified DNA as template each component of the PCR reaction was adjusted for the optimum amplification of the DNA sequence. Optimum specific amplification resulted when the primer annealing temperature was 60 degrees C. The gene fragment was amplifiable in 25 different Brucella species and strains. To test the specificity of the reaction, DNA extracted from 17 micro-organisms possibly associated with cattle were tested. No amplification was observed. The sensitivity of the reaction was determined with different concentrations of genomic Brucella strain 19 DNA. As little as 0.1 pg DNA (less than 100 brucella cells) could be detected. The specificity and sensitivity of PCR combined with its simplicity and speed suggests the potential of this technique for routine diagnosis of brucellosis.     

猪PAC克隆的微卫星DNA分离研究

利用(CA)8核心序列,设计锚定引物,采用直接测序法,从单个猪PAC克隆中分离到一个新微卫星DNA。根据该微卫星DNA的侧翼序列,设计了专一引物,在8个猪种40个个体中检测到3个等位基因,片段长度分别为305 bp、307 bp和309 bp。3种等位基因纯合子个体的PCR产物序列分析表明,这3种等位基因分别有12、13和14次CA双核苷酸重复。 Abstract:A novel microsatellite DNA was isolated from a single porcine PAC clone by sequencing the PAC clone directly with (CA)n repeat motif anchored primer.The specific primer pairs flanking the (CA)n repeat region were used to amplify the genomic DNA of 40 individuals from 8 pig breeds,which detected three alleles with the fragment length of 305 bp,307 bp and 309 bp.The PCR product sequencing results of homozygous animals representing three alleles revealed that those three alleles contained 12,13 and 14 CA dinucleotide repeats respectively.