Transcriptor Reverse Transcriptase – the core component of the kit – is a recombinant reverse transcriptase expressed in . The enzyme has RNA-directed DNA polymerase activity, DNA-dependent DNA polymerase activity, unwinding activity, and RNase H activity that degrades RNA in RNA:DNA hybrids. The latter circumvents the need to perform an additional time-consuming RNase H incubation step after reverse transcription. This shortens the reaction time and reduces costs.
Transcriptor Reverse Transcriptase is recommended for RT-PCR because of its high sensitivity in combination with high thermostability: the enzyme synthesizes long cDNA products (up to 14 kb) and can be used at temperatures up to +65°C. Due to its thermostability, Transcriptor Reverse Transcriptase is recommended for GC-rich templates with high secondary structure, without the need to include additives in the reaction.
The kit provides all reagents required for first-strand cDNA synthesis reactions. For priming, three different primer systems can be used. Two cDNA synthesis primers are provided with the kit: random hexamer primers and an anchored-oligo(dT)18 primer. The latter is designed to bind at the beginning of the poly(A) tail to generate full-length cDNAs and to prevent priming from internal sites of the poly(A) tail. The 5' ends of long mRNAs are often underrepresented; therefore, this priming method is preferred for most applications. The use of random hexamer primers enables priming throughout the length of RNA for uniform representation of all RNA sequences and allows reverse transcription of RNAs that do not carry a poly(A) tail.
Thermostable Protector RNase Inhibitor is included in the kit to protect RNA from degradation at high reaction temperatures.
Mitochondrial DNAs (mtDNAs) vary extensively in size and gene content across diverse eukaryotic groups, while those of animals (Metazoa), however are relatively more uniform (). As of May 2006, there are 13 nematode mtDNA sequences in the GenBank (. Although nematode mitochondrial genome sequence data available thus far conforms more or less with current generalizations concerning metazoan mtDNAs, the genomes do contain some unique features and common characteristics. These include 12 protein-coding genes (lacking the gene, except for in which it is also encoded), the lack of either a DHU or TC arm in the tRNA secondary structure and apparent unidirectional transcription, where all genes occur on the same strand (see for details), with the exception of () and (He et al., unpublished) where some genes would be transcribed from the opposite strand. Convergent evolution (not derived from a common ancestry) of gene rearrangement is generally known to be rare (). Therefore, comparative analysis of the mitochondrial genome information (e.g., gene arrangement, nucleotide and amino acid sequences) has often been used as a reliable tool for resolving the phylogenetic relationships in a large number of diverse animal groups with ancient evolutionary origins (; ; ). The mitochondrial genome of revealed that its organization more closely resembles that of the coelomate metazoans than that of its presumed closest relatives, the secernentean nematodes (). Furthermore, is the only nematode species known in which mtDNA includes a putative atp8 gene (as discussed above), bringing the total of recognized mtDNA genes to 37 genes, which is typical of most metazoan mtDNA ().
Is a random primer or oligo(dT) better ..
Until recently the approach taken to study involved mainly characterization of individual genes of interest. In 2003, a genomic approach was initiated as an antecedent to more complete nuclear genome sequencing (). The approach involves use of expressed sequence tags (ESTs) obtained from sampling 3 cDNA libraries generated from three life stages of : adult worm (AD), mature muscle larvae (ML) and immature L1 larvae (immL1, also known as newborn larvae). The analysis of the 10,130 ESTs identified a conservative estimate of 3,262 unique genes. Based on genomic information from (), this number represents 17% (3,262/19,552) of all genes. The GC content for protein coding exons was 39% versus 43% for . According to this study, 56% of the EST clusters had homology to proteins from other species, while 44% were placed in the category of ‘novel’ proteins. Furthermore, 82% of the clusters with homology (1592/1942) had homology to (or 46% of all clusters). The most recent meta-analysis of the transcriptome of the Phylum Nematoda () reported similar results (45%), and expanding the analysis to species beyond nematodes, identified a similar portion of the ESTs sharing homology with the fruit fly . Hence, ESTs common to and are not necessarily specific to nematodes but may be conserved among diverse taxonomic groups of invertebrates. is often thought of, and therefore used as, a protopypical nematode because of its usefulness to serve as a model to study biological processes. However, the results from the more extensive single-species analysis () and more broad Phylum-related analysis () highlight the great phylogenetic distance of from other nematodes, which makes the extrapolation from the biology of to challenging. However, comparative genomic approaches using both nematodes may be useful to identify molecular features shared between these two widely disparate species that reflect ancestral features found in many nematodes. Furthermore, the authors segregated the identified genes in multiple biological dimensions including functional, developmental and phylogenetic categories. Nematode genes can now be cross-referenced to gain insight on higher order associations. Observations agreed with and extended information on previously described genes and gene families, providing expectations that information on newly discovered genes will have similar value. The data identified sets of predicted proteins which may define differences in metabolism and molecular interactions that exist among the stages investigated. Evidence of substantial gene families in relation to previously identified antigen genes was especially instructive, as was elucidation of numerous and diverse predicted proteinase genes. The dataset is potentially very useful for proteomics methods (as described in ) to identify parasite proteins that occur in specific compartments, such as host muscle nuclei and parasite excretory-secretory products and the external cuticular surface. The observations made provide strong rationale to gain a more complete assessment of genes expressed among stages. From a phylogenetic perspective, adenophorean orders that comprise clade I, such as the Trichocephalida (), Mermithida, Dorylaimida, and Mononchida, remain largely unexplored territory for genomic studies. This initial analysis of expressed genes in confirms an ancient divergence of clade I nematodes from those of other clades and provides an entry point toward a deep understanding of the phylum Nematoda at the molecular level. These findings have stimulated a nuclear genome sequencing project for with this goal in mind, discussed in .
cDNA Synthesis; cDNA Synthesis Kits; ..
The reaction requires primers, which can be either oligodT (annealing to polyA tails of mRNA) or random hexamers; the kit used here contains both kinds); nucleotides for DNA synthesis (dNTPs); MgC2 and buffers required by the enzyme, which are all present in the 5x Reaction Mix.
Transcriptor First Strand cDNA Synthesis Kit
Three types of primers can be used for RT reaction: oligo (dT) primers, random (hexamer) primers and gene specific primers with each having its pros and cons.
cDNA synthesis using superscript II - Untergasser
The Transcriptor First Strand cDNA Synthesis Kit is designed to reverse transcribe RNA (mRNA, total RNA, viral RNA, and -transcribed RNA) from a variety of sources for the following applications: