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An economical and efficient method of post reaction cleanup of labeled dye terminator sequencing products
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Sanger dideoxy DNA sequencing method is the most popular and simple DNA sequencing method and the most economic method available in the market as per as the throughput is concerned. The good quality of sequences depends not only on the quality of the template but also on efficient removal of unincorporated fluorescent labeled dideoxy nucleotides (ddNTPs). The present study reports an economical and efficient method of post reaction cleanup of labeled unincorporated ddNTPs. The efficiency of the method was compared with different methods. EDTA-Isopropanol method gave very good sequence compared to other methods used in the present study. The EDTA-Isopropanol method gave good quality (QV≥ 20) sequence of 86 bases out of 120 bases of initial region of the sequence which covers both dye blobs. The enhancement of the quality of sequences especially at the initial region of 120 bases not only makes the analysis part easy but also reduces the use of number of primers. Subsequently, reducing the cost of sequencing.
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Ethambutol [EMB: dextro-2.2′-(ethylenediimino)-di-l- butanol], a frontline antituberculosis drug, targets the mycobacterial cell wall. Genetic events resulting in structural mutations at the embB locus have been pro- posed as a major mechanism for EMB resistance in Mycobacterium tuberculosis. In this study, mutations in the embB locus of Indian isolates and their correla- tion with the MIC levels in M. tuberculosis have been investigated. We have sequenced and analysed 260 bp of the embB gene region containing the hotspot asso- ciation with EMB-resistance isolates of M. tuberculosis in 39 EMB-resistant and 13 susceptible clinical iso- lates. Out of 39 resistant isolates, 17 (44%) had muta- tion in the embB locus at codon 306, whereas the remaining 22 (56%) resistant and 13 sensitive isolates did not have any mutation in this region. Five differ- ent substitution mutations of ATG(Met) in the resis- tant strains were: GTG (Val) 41%, ATA (Ile) 29%, ATC (Ile) 18%, TTG (Leu) 6% and CTG (Leu) 6%. All these mutations at codon 306 were associated with a high degree of ethambutol-resistant phenotype. Results suggest that the embB306 mutations might prove to be one of useful markers for detection of ethambutol re- sistance in isolates of M. tuberculosis in India. Lack of mutation, however, does not rule out ethambutol resis- tance.
We have developed a quick and low-cost genomic DNA extraction protocol from yeast cells for PCR-based applications. This method does not require any enzymes, hazardous chemicals, or extreme temperatures, and is especially powerful for simultaneous analysis of a large number of samples. DNA can be efficiently extracted from different yeast species (Kluyveromyces lactis, Hansenula polymorpha, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris, and Saccharomyces cerevisiae). The protocol involves lysis of yeast colonies or cells from liquid culture in a lithium acetate (LiOAc)-SDS solution and subsequent precipitation of DNA with ethanol. Approximately 100 nanograms of total genomic DNA can be extracted from 1 × 10(7) cells. DNA extracted by this method is suitable for a variety of PCR-based applications (including colony PCR, real-time qPCR, and DNA sequencing) for amplification of DNA fragments of ≤ 3500 bp.
A high-throughput DNA sequencing method that generated high quality data was developed. A frame fashioned from a standard agarose gel combined with 0.1%-0.2% low-melting point (LMP) agarose gel was used to isolate the PCR product of interest. Collected PCR products were centrifuged without any reagents and the supernatants were directly used for a sequencing reaction. This method is simple and labor efficient, provides high quality sequences at a low cost, and bypasses problems with impure PCR products. This technique has been used for single nucleotide polymorphism (SNP) discovery in Populus angustifolia trees.
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
- R Das
- K Katoch
- G P S Jadauan
- P Gupta
- V D Sharma
- B Malhotra
- A Garg
Das R., Katoch, K., Jadauan, G.P.S., Gupta, P., Sharma, V.D., Malhotra, B., Garg A. et al. (2006): Studies on
Mutations in Embb Locus In Indian Clinical Isolates of Mycobacterium Tuberculosis Having High Degree
of Ethambutol Resistance. Curr. Sci. 91: 293-295.