Forensic botany II,DNA barcode for land plants: Which markers after the international agreement?

The ambitious idea of using a short piece of DNA for large-scale species identification (DNA barcoding) is already a powerful tool for scientists and the application of this standard technique seems promising in a range of fields including forensic genetics. While DNA barcoding enjoyed a remarkable success for animal identification through cytochrome c oxidase I (COI) analysis, the attempts to identify a single barcode for plants remained a vain hope for a longtime. From the beginning, the Consortium for the Barcode of Life (CBOL) showed a lack of agreement on a core plant barcode, reflecting the diversity of viewpoints. Different research groups advocated various markers with divergent set of criteria until the recent publication by the CBOL–Plant Working Group. After a four-year effort, in 2009 the International Team concluded to agree on standard markers promoting a multilocus solution (rbcL and matK), with 70–75% of discrimination to the species level. In 2009 our group firstly proposed the broad application of DNA barcoding principles as a tool for identification of trace botanical evidence through the analysis of two chloroplast loci (trnH-psbA and trnL-trnF) in plant species belonging to local flora. Difficulties and drawbacks that were encountered included a poor coverage of species in specific databases and the lack of authenticated reference sequences for the selected markers. Successful preliminary results were obtained providing an approach to progressively identify unknown plant specimens to a given taxonomic rank, usable by any non-specialist botanist or in case of a shortage of taxonomic expertise. Now we considered mandatory to update and to compare our previous findings with the new selected plastid markers (matK + rbcL), taking into account forensic requirements.Features of all the four loci (the two previously analyzed trnH-psbA + trnL-trnF and matK + rbcL) were compared singly and in multilocus solutions to assess the most suitable combination for forensic botany.Based on obtained results, we recommend the adoption of a two-locus combination with rbcL + trnH-psbA plastid markers, which currently best satisfies forensic needs for botanical species identification.     

Cloning and sequence analysis of β-actin gene from Aedes albopictus （Diptera： Culicidae）

Objective: To obtain the complete β-actin gene from Aedes albopictus. Methods: Total RNA was extracted from C6/36 cells. Degenerate primers were designed based on the β-actin sequences of An. gambiae, Ae. aegypti, Cx. pipiens pallens and D.melanogaster. By RT-PCR, the product was amplified, purified, cloned into the pGT vector and sequenced. The β-actin sequence was aligned and phylogenetically analyzed by the BLAST program and the CLUSTAL W program. Results: A sequence of 1132 bp including an open reading frame of 1131 bp was obtained (GenBank DQ657949). The deduced protein had 376 amino acids.Aligned to SWISS-PROT, it exhibited a high level of identity with β-actins from Anopheles, Drosophila and Culex at the amino acid sequence level. Phylogenetic analysis indicated that Ae. albopictus β-actin was much more homologous with invertebrate β-actin than with vertebrate β-actin. Conclusion: The gene may be used as the internal control in the experiments of Ae. albopictus.     

GenBank数据库和PubMed数据库中序列数据信息检索比较

通过对GenBank数据库和PubMed数据库的数据来源、检索界面和检索结果等的对比分析,发现2个检索库检索的序列数据信息存在差别,GenBank数据库检索结果和检准率均高于PubMed数据库。     

硕大利什曼原虫无鞭毛体蛋白的基因克隆与序列分析

[目的 ]克隆硕大利什曼原虫无鞭毛体蛋白 (amastin)的编码基因序列。 [方法 ]应用核苷酸序列数据库 (GenBank)和表达序列末端片段数据库 (dbEST)的计算机检索与DNA文库的杂交筛选方法。 [结果 ]从dbEST数据库中获得一段 30 9nt的来源于硕大利什曼原虫的基因片段 ,据此设计探针 ,筛选硕大利什曼原虫的DNA文库 ,获得硕大利什曼原虫无鞭毛体蛋白的编码基因。其开放读码框架由 5 5 2个核苷酸组成 ,编码产物由183个氨基酸残基组成。序列分析表明 ,硕大利什曼原虫与锥虫无鞭毛体蛋白一级结构的同源性为 2 3 5 %。 [结论 ]克隆的基因系硕大利什曼原虫表面蛋白编码基因 ,即无鞭毛体蛋白的编码基因。     

小鼠肝再生增强因子cDNA的克隆化与序列分析

目的克隆小鼠肝再生增强因子（ＡＬＲ）的ｃＤＮＡ，并对其同源性序列进行检索与分析。方法以 人和大鼠的ＡＬＲ ｃＤＮＡ作为参考，以ＢＬＡＳＴ为检索途径，对美国国立生物工程学信息中心（ＮＣＢＩ）建立的核苷酸 序列数据库（ＧｅｎＢａｎｋ）进行检索，检出了小鼠ＡＬＲ基因组ＤＮＡ，ＧｅｎＢａｎｋ收录号为Ｕ４０４９４。根据大鼠、人ＡＬＲ 与小鼠ＡＬＲ同源性原则，设计了小鼠ＡＬＲ的特异性引物，以聚合酶链反应（ＰＣＲ）技术扩增获得了小鼠ＡＬＲ ｃＤＮＡ 片段，测序并进行核苷酸序列以及编码产物的氨基酸残基序列的同源性分析。结果获得了小鼠ＡＬＲ ｃＤＮＡ基 因片段，全长为５５９个核苷酸（ｎｔ），编码区为３７５ｎｔ，编码由１２５个氨基酸残基组成的多肽，计算分子量为１．５ × １０~４， 与人、大鼠及酵母ＥＲＶ１基因高度同源。结论小鼠ＡＬＲ ｃＤＮＡ编码一种酵母ＥＲＶ１的同源蛋白质。     

Sequence-based genotyping HPV L1 DNA and RNA transcripts in clinical specimens

We developed a direct sequence-based genotyping method to detect single and multiple HPV L1 DNA and RNA types in genital and dermatological specimens. Our method couples PCR amplification of a highly conserved HPV L1 segment using a broad spectrum-generic primer cocktail mix with automated sequencing of amplified PCR products, followed by GenBank sorting of sequencing data. We genotyped 5 skin and 30 cervical HPV DNA-positive specimens using this method and established its first experimentally derived working cutoff value with the aid of commercial hybridization-based techniques. We suggest that sequence-based genotyping of appropriately amplified DNA and RNA products may serve as a primary HPV detection method in dermatological specimens. It can be applied as an all-purpose genotyping method for rare HPV types not detectable by commercial hybridization-based techniques and for sorting multiple HPV infections by order of prevalence.     

Establishment of a molecular tool for blood meal identification in Malaysia

Objective

To establish a polymerase chain reaction (PCR) technique based on cytochrome b (cytb) gene of mitochondria DNA (mtDNA) for blood meal identification.

Methods

The PCR technique was established based on published information and validated using blood sample of laboratory animals of which their whole gene sequences are available in GenBank. PCR was next performed to compile gene sequences of different species of wild rodents. The primers used were complementary to the conserved region of the cytb gene of vertebrate''s mtDNA. A total of 100 blood samples, both from laboratory animals and wild rodents were collected and analyzed. The obtained unknown sequences were compared with those in the GenBank database using BLAST program to identify the vertebrate animal species.

Results

Gene sequences of 11 species of wild animals caught in 9 localities of Peninsular Malaysia were compiled using the established PCR. The animals involved were Rattus (rattus) tanezumi, Rattus tiomanicus, Leopoldamys sabanus, Tupaia glis, Tupaia minor, Niviventor cremoriventor, Rhinosciurus laticaudatus, Callosciurus caniseps, Sundamys muelleri, Rattus rajah and Maxomys whiteheadi. The BLAST results confirmed the host with exact or nearly exact matches (>89% identity). Ten new gene sequences have been deposited in GenBank database since September 2010.

Conclusions

This study indicates that the PCR direct sequencing system using universal primer sets for vertebrate cytb gene is a promising technique for blood meal identification.     

The TP53 mutation, R337H, is associated with Li-Fraumeni and Li-Fraumeni-like syndromes in Brazilian families

A TP53 germline mutation, R337H, has been previously described in children from southern Brazil with adrenocortical tumours but no documented familial history of other cancers. Here, we have screened for TP53 mutation 45 Brazilian unrelated individuals with family histories fulfilling the clinical definitions of Li-Fraumeni (LFS) or Li-Fraumeni-like (LFL) syndromes. Mutations were found in 13 patients (28.9%), including six (46.1%) R337H mutations, and four novel germline mutations (V173M, V197M, G244D and IVS6+1G>T). Families with the R337H mutation presented a wide spectrum of tumours, including breast cancers (30.4%), brain cancers (10.7%), soft tissue sarcomas (10.7%) and adrenocortical carcinomas (8.9%). Testing of 53 Brazilian subjects with no cancer history showed that R337H was not a common polymorphism in that population. Moreover, loss of heterozygocity with retention of the R337H allele was observed in a breast adenocarcinoma, supporting a role for this mutation in breast tumorigenesis. These results show that the TP53 R337H germline mutation predisposes to a larger spectrum of tumours, similar to the one reported for other TP53 mutations.     

集成化数据库检索系统Entrez的开发与应用

Entrez是美国生物技术信息中心开发研制的集成化数据库检索系统,用户可以检索和浏览与基因库(GenBank)链接的所有数据库.本文主要探讨了新加入到Entrez数据检索系统的PubMed以及具有特殊功能的邻接系统(Neighboring System).