DNA

Evaluation of methods of DNA extraction from Staphylococcus aureus in milk for use in real-time PCR

A. G. Dibbern, Botaro, B. G., Viziack, M. P., Silva, L. F. P., and Santos, M. V., Evaluation of methods of DNA extraction from Staphylococcus aureus in milk for use in real-time PCR, vol. 14, pp. 227-233, 2015.

The aim of this study was to evaluate the repeatability and performance of 4 methods of extracting DNA from Staphylococcus aureus (SAU) and the gene encoding bovine mitochondrial cytochrome B (BMCB) in milk samples from cows with subclinical mastitis for use in amplification by real-time polymerase chain reaction. Two milk samples were obtained from cows naturally infected with S. aureus and subjected to the following extraction methods: Qiagen DNA extraction kit; Axyprep DNA extraction kit; in silica column boil and in silica column method.

Molecular identification of phosphate-solubilizing native bacteria isolated from the rhizosphere of Prosopis glandulosa in Mexicali valley

L. Moreno-Ramírez, González-Mendoza, D., Cecena-Duran, C., and Grimaldo-Juarez, O., Molecular identification of phosphate-solubilizing native bacteria isolated from the rhizosphere of Prosopis glandulosa in Mexicali valley, vol. 14, pp. 2793-2798, 2015.

One of the main limitations in intensive crop production in Northwestern Mexico is the dependence on the use of phosphate fertilizer. In this study, we isolated indigenous microorganisms with phosphate solubilization capacities from mesquite (Prosopis glandulosa) present in the Mexicali valley. In total, 4 bacteria were isolated from the rhizosphere of mesquite, including ICA01, ICA02Ba, ICA03Bs, and ICA04Ma. The bacterial isolates were identified based on their phenotypic and 16S rRNA gene sequencing data to be Acinetobacter calcoaceticus.

A multiplex panel of short-amplicon insertion-deletion DNA polymorphisms for forensic analysis

V. R. D. Santos, Pena, H. B., and Pena, S. D. J., A multiplex panel of short-amplicon insertion-deletion DNA polymorphisms for forensic analysis, vol. 14, pp. 2947-2952, 2015.

We have previously developed a panel of 40 insertion-deletion (INDEL) human DNA polymorphisms that was proven to ad­equately cover the span of global human genetic diversity. The panel was found to have very low matching probabilities with respect to both the global and Brazilian populations. To optimize the panel for application with degraded DNA samples, which are commonly encountered in fo­rensic analysis, we have significantly reduced the amplicon size of the INDELs and developed a new multiplex panel.

A non-destructive genotyping system from a single seed for marker-assisted selection in watermelon

G. Meru, McDowell, D., Waters, V., Seibel, A., Davis, J., and McGregor, C., A non-destructive genotyping system from a single seed for marker-assisted selection in watermelon, vol. 12, pp. 702-709, 2013.

Genomic tools for watermelon breeding are becoming increasingly available. A high throughput genotyping system would facilitate the use of DNA markers in marker-assisted selection. DNA extraction from leaf material requires prior seed germination and is often time-consuming and cost prohibitive. In an effort to develop a more efficient system, watermelon seeds of several genotypes and various seed sizes were sampled by removing ⅓ or ½ sections from the distal ends for DNA extraction, while germinating the remaining proximal parts of the seed.

Development and characterization of microsatellite loci in a threatened marine fish, Cheilinus undulatus (humphead wrasse)

J. Hu, Zhu, X. P., Luo, J., Yin, S. W., Peng, Y. H., Hu, Y. L., and Zhu, F., Development and characterization of microsatellite loci in a threatened marine fish, Cheilinus undulatus (humphead wrasse), vol. 12, pp. 2633-2636, 2013.

Cheilinus undulatus (humphead wrasse) is a marine fish distributed widely throughout the tropical Indo-Pacific. It has been listed as vulnerable in the IUCN Red Data Book and in CITES Appendix II four times. Fifteen microsatellite loci were isolated and characterized for this species. The number of alleles ranged from 3 to 15 per locus, and the observed and expected heterozygosity ranged between 0.0323-0.7742 and 0.2597-0.8773, respectively. The polymorphism information content ranged from 0.2353-0.8520.

Efficient human paternity testing with a panel of 40 short insertion-deletion polymorphisms

J. R. Pimenta and Pena, S. D. J., Efficient human paternity testing with a panel of 40 short insertion-deletion polymorphisms, vol. 9, pp. 601-607, 2010.

We developed a panel of 40 multiplexed short insertion-deletion (indel) polymorphic loci with widespread chromosomal locations and allele frequencies close to 0.50 in the European population. We genotyped these markers in 360 unrelated self-classified White Brazilians and 50 mother-child-probable father trios with proven paternity. The average heterozygosity (gene diversity) per locus was 0.48, and the combined probability of identity (matching probability) for the 40-locus set was 3.48 x 10-17. The combined power of exclusion of the indel panel was 0.9997.

Optimization of DNA extraction from seeds and fresh leaf tissues of wild marigold (Tagetes minuta) for polymerase chain reaction analysis

I. Shahzadi, Ahmed, R., Hassan, A., and Shah, M. M., Optimization of DNA extraction from seeds and fresh leaf tissues of wild marigold (Tagetes minuta) for polymerase chain reaction analysis, vol. 9, pp. 386-393, 2010.

Tagetes, a genus of flowering marigolds in the family Asteraceae (Compositeae), is reported to be a medicinal plant with hypotensive, spasmolytic, anti-inflammatory, antimicrobial, and antifungal properties. Tagetes minuta characteristically contains high concentrations of essential oils, flavonoids, polyphenols, and polysaccharides that interfere with DNA, causing erroneous or no PCR products. We tested and modified various standard protocols in an effort to isolate high-quality DNA from different plant tissues of T. minuta.

Usefulness of direct sequencing of pooled DNA for SNP identification and allele-frequency determination compatible with a common disease/common variant hypothesis

M. K. Kim, Nam, T. S., Choi, K. H., Jang, S. Y., Kim, Y. O., and Lee, M. C., Usefulness of direct sequencing of pooled DNA for SNP identification and allele-frequency determination compatible with a common disease/common variant hypothesis, vol. 9, pp. 772-779, 2010.

We examined the efficiency of direct sequencing of pooled DNA for developing common single nucleotide polymorphisms (SNPs) and its accuracy for estimating allele frequencies. A pool of 200 control DNAs was established and was used for developing SNPs and estimating minor allele frequencies (MAF). The sensitivity of the pooled DNA method for successfully detecting an SNP with an MAF >0.01 listed in the database was approximately 0.7; it was particularly efficient for detecting SNPs with MAF >0.1, which is compatible with the common disease/common variant hypothesis.

Worldwide diversity of the Y-chromosome tetra-local microsatellite DYS464

F. S. G. Kehdy and Pena, S. D. J., Worldwide diversity of the Y-chromosome tetra-local microsatellite DYS464, vol. 9, pp. 1525-1534, 2010.

Of all DNA markers on the human Y-chromosome, the tetra-local Y-linked microsatellite DYS464 is the most polymorphic. We genotyped DYS464 in 677 male samples collected worldwide, maintained in the HGDP-CEPH Human Genome Diversity Cell Line Panel. Fourteen different alleles were found, with allele lengths varying from 9 to 23 repeats. One hundred and seventy-five different genotypes were detected, of which 90 appeared to be continent-specific. The region with the highest percentage of unique genotypes was Africa.

Comparison of three methods of DNA extraction from peripheral blood mononuclear cells and lung fragments of equines

E. M. Santos, Paula, J. F. R., Motta, P. M. C., Heinemann, M. B., Leite, R. C., Haddad, J. P. A., Del Puerto, H. L., and Reis, J. K. P., Comparison of three methods of DNA extraction from peripheral blood mononuclear cells and lung fragments of equines, vol. 9. pp. 1591-1598, 2010.

We compared three different protocols for DNA extraction from horse peripheral blood mononuclear cells (PBMC) and lung fragments, determining average final DNA concentration, purity, percentage of PCR amplification using β-actin, and cost. Thirty-four samples from PBMC, and 33 samples from lung fragments were submitted to DNA extraction by three different protocols. Protocol A consisted of a phenol-chloroform and isoamylic alcohol extraction, Protocol B used alkaline extraction with NaOH, and Protocol C used the DNAzol® reagent kit.

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