Quantitative Analysis of Mutant Subclones in Chronic Myeloid Leukemia: Comparison of Different Methodological Approaches.

Identification and quantitative monitoring of mutant BCR-ABL1 subclones displaying resistance to tyrosine kinase inhibitors (TKIs) have become important tasks in patients with Ph-positive leukemias. Different technologies have been established for patient screening. Various next-generation sequencing (NGS) platforms facilitating sensitive detection and quantitative monitoring of mutations in the ABL1-kinase domain (KD) have been introduced recently, and are expected to become the preferred technology in the future. However, broad clinical implementation of NGS methods has been hampered by the limited accessibility at different centers and the current costs of analysis which may not be regarded as readily affordable for routine diagnostic monitoring. It is therefore of interest to determine whether NGS platforms can be adequately substituted by other methodological approaches. We have tested three different techniques including pyrosequencing, LD (ligation-dependent)-PCR and NGS in a series of peripheral blood specimens from chronic myeloid leukemia (CML) patients carrying single or multiple mutations in the BCR-ABL1 KD. The proliferation kinetics of mutant subclones in serial specimens obtained during the course of TKI-treatment revealed similar profiles via all technical approaches, but individual specimens showed statistically significant differences between NGS and the other methods tested. The observations indicate that different approaches to detection and quantification of mutant subclones may be applicable for the monitoring of clonal kinetics, but careful calibration of each method is required for accurate size assessment of mutant subclones at individual time points.

Molecular cloning of the alpha subunit of complement component C8 (CpC8α) of whitespotted bamboo shark (Chiloscyllium plagiosum).

Complement-mediated cytolysis is the important effect of immune response, which results from the assembly of terminal complement components (C5b-9). Among them, α subunit of C8 (C8α) is the first protein that traverses the lipid bilayer, and then initiates the recruitment of C9 molecules to form pore on target membranes. In this article, a full-length cDNA of C8α (CpC8α) is identified from the whitespotted bamboo shark (Chiloscyllium plagiosum) by RACE. The CpC8α cDNA is 2183 bp in length, encoding a protein of 591 amino acids. The deduced CpC8α exhibits 89%, 49% and 44% identity with nurse shark, frog and human orthologs, respectively. Sequence alignment indicates that the C8α is well conserved during the evolution process from sharks to mammals, with the same modular architecture as well as the identical cysteine composition in the mature protein. Phylogenetic analysis places CpC8α and nurse shark C8α in cartilaginous fish clade, in parallel with the teleost taxa, to form the C8α cluster with higher vertebrates. Hydrophobicity analysis also indicates a similar hydrophobicity of CpC8α to mammals. Finally, expression analysis revealed CpC8α transcripts were constitutively highly expressed in shark liver, with much less expression in other tissues. The well conserved structure and properties suggests an analogous function of CpC8α to mammalian C8α, though it remains to be confirmed by further study.

Molecular characterization and expression analysis of complement component C9 gene in the whitespotted bambooshark, Chiloscyllium plagiosum.

Complement system is known as highly sophisticated immune defense mechanism for antigen recognition as well as effector functions. Activation of the terminal pathway of the complement system leads to the assembly of terminal complement complexes (C5b-9), which induces the characteristic complement-mediated cytolysis. The lytic activity of shark complement involves functional analogues of mammalian C8 and C9. In this article, a full-length cDNA of C9 (CpC9) is identified from cartilaginous species, the whitespotted bambooshark, Chiloscyllium plagiosum by RACE. The CpC9 cDNA is 2263 bp in length, encoding a protein of 603 amino acids, which shares 42% and 43% identity with human and Xenopus C9 respectively. Through sequence alignment and comparative analysis, the CpC9 protein was found well conserved, with the typical modular architecture in TCCs and nearly unanimous cysteine composition from fish to mammal. Phylogenetic analysis places it in a clade with C9 orthologs in higher vertebrate and as a sister taxa to the Xenopus. Expression analysis revealed that CpC9 is constitutively highly expressed in shark liver, with much less or even undetectable expression in other tissues; demonstrating liver is the primary tissue for C9synthesis. To sum up, the structural conservation and distinctive phylogenetics might indicate the potentially vital role of CpC9 in shark immune response, though it remains to be confirmed by further study.

Eighteen years of molecular genotyping the hemophilia inversion hotspot: from southern blot to inverse shifting-PCR.

The factor VIII gene (F8) intron 22 inversion (Inv22) is a paradigmatic duplicon-mediated rearrangement, found in about one half of patients with severe hemophilia A worldwide. The identification of this prevalent cause of hemophilia was delayed for nine years after the F8 characterization in 1984. The aim of this review is to present the wide diversity of practical approaches that have been developed for genotyping the Inv22 (and related int22h rearrangements) since discovery in 1993. The sequence- Southern blot, long distance-PCR and inverse shifting-PCR-for Inv22 genotyping is an interesting example of scientific ingenuity and evolution in order to resolve challenging molecular diagnostic problems.

Study on construction of cDNA libraries from rat normal liver and regeneration liver with smart technique.

The aim of the study is to construct cDNA libraries from the normal liver and regeneration liver of rat by SMART (switching mechanism at 5' end of RNA transcript) technique and analyze their quality. The total RNA was separated from the normal liver and regeneration liver of rat and the frist-strand cDNA was synthesized through reverse transcription by a modified oligo (dT) primer (contained sfi IB site) while the SMART oligonucleotide (contained sfi IA site) was utilized as a template so that the first-strand cDNA could be extended over the 5' end of mRNA. The double-strand cDNA was amplified by LD-PCR (long-distance PCR) with the above two primers and then digested by sfi I (IA & IB) restriction, enzyme. After cDNA size fractionation through Chroma Spin 400 column, the double-strand cDNA was ligated into the sfi I-digested lambda TripIEx2 vector and then the recombinant DNA was packagedin vitro. The unamplified rat normal liver cDNA library consists of 1.3×10(7) pfu/ml, and regeneration liver cDNA library consists of 1.6×10(7) pfu/ml in which the percentage of recombinant clones both are about 100%. Through testing, the high quality cDNA libraries containing full-length cDNA of rat normal liver and regeneration liver have been constructed. The titer of the amplified cDNA library is 4.5×10(10) pfu/ml and 3.6×10(10) pfu/ml. the average exogenous inserts of the recombinants both are about 1.5 kb. These results show that the normal liver and regeneration liver of rat cDNA libraries both have an excellent quality and lay solid foundation to study liver functions and the mechanism of liver regeneration.

Study on construction of cDNA library of the treated changliver cell and quality analysis.

The study aims to construct cDNA library of Changliver cell by SMART (switching mechanism at 5' end of RNA transcript) technique and analyze its quality. cDNA of Changliver cell was made with RT-PCR and LD-PCR (long-distance PCR), the cDNA library was constructed with SMART cDNA library construction kit. Through testing, the high quality cDNA library containing whole long cDNA of Changliver cell had been constructed. The titer of the amplified cDNA library was 4.5 × 10(10) pfu/ml and the average exogenous inserts of the recombinants is 1.5 kb. These results showed that the Changliver cell cDNA library had an excellent quality and lay foundation for screening whole long cDNA of related genes.