ABSTRACT
Reverse transcriptases (RTs) are a standard tool in both fundamental studies and diagnostics. RTs should possess elevated temperature optimum, high thermal stability, processivity and tolerance to contaminants. Here, we constructed a set of chimeric RTs, based on the combination of the Moloney murine leukaemia virus (M-MuLV) RT and either of two DNA-binding domains: the DNA-binding domain of the DNA ligase from Pyrococcus abyssi or the DNA-binding Sto7d protein from Sulfolobus tokodaii. The processivity and efficiency of cDNA synthesis of the chimeric RT with Sto7d at the C-end are increased several fold. The attachment of Sto7d enhances the tolerance of M-MuLV RT to the most common amplification inhibitors: NaCl, urea, guanidinium chloride, formamide, components of human whole blood and human blood plasma. Thus, fusing M-MuLV RT with an additional domain results in more robust and efficient RTs.
Subject(s)
DNA-Binding Proteins/metabolism , Drug Resistance, Viral , Moloney murine leukemia virus/enzymology , RNA-Directed DNA Polymerase/metabolism , Recombinant Fusion Proteins/metabolism , Reverse Transcriptase Inhibitors/pharmacology , Sulfolobus , DNA, Complementary/biosynthesis , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , Enzyme Stability , Ions , Protein Denaturation , Protein Domains , RNA-Directed DNA Polymerase/chemistry , RNA-Directed DNA Polymerase/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Sequence Homology, Amino Acid , TemperatureABSTRACT
At the moment, one of the actual trends in medical diagnostics is a development of methods for practical applications such as point-of-care testing, POCT or research tools, for example, whole genome amplification, WGA. All the techniques are based on using of specific DNA polymerases having strand displacement activity, high synthetic processivity, fidelity and, most significantly, tolerance to contaminants, appearing from analysed biological samples or collected under purification procedures. Here, we have designed a set of fusion enzymes based on catalytic domain of DNA polymerase I from Geobacillus sp. 777 with DNA-binding domain of DNA ligase Pyrococcus abyssi and Sto7d protein from Sulfolobus tokodaii, analogue of Sso7d. Designed chimeric DNA polymerases DBD-Gss, Sto-Gss and Gss-Sto exhibited the same level of thermal stability, thermal transferase activity and fidelity as native Gss; however, the processivity was increased up to 3-fold, leading to about 4-fold of DNA product in WGA which is much more exiting. The attachment of DNA-binding proteins enhanced the inhibitor tolerance of chimeric polymerases in loop-mediated isothermal amplification to several of the most common DNA sample contaminants-urea and whole blood, heparin, ethylenediaminetetraacetic acid, NaCl, ethanol. Therefore, chimeric Bst-like Gss-polymerase will be promising tool for both WGA and POCT due to increased processivity and inhibitor tolerance.
Subject(s)
DNA Polymerase I/metabolism , Geobacillus/enzymology , Protein Engineering/methods , Recombinant Fusion Proteins/genetics , Catalytic Domain , Cloning, Molecular , DNA/metabolism , DNA Polymerase I/antagonists & inhibitors , DNA Polymerase I/genetics , Enzyme Inhibitors/pharmacology , Genome, Human , Geobacillus/genetics , Geobacillus stearothermophilus/enzymology , Geobacillus stearothermophilus/genetics , Humans , Nucleic Acid Amplification Techniques/methods , Protein Stability , Pyrococcus abyssi/genetics , Recombinant Fusion Proteins/metabolism , Sulfolobus/geneticsABSTRACT
The aim of this study was to implement massive parallel sequencing (MPS) technology in clinical genetics testing. We developed and tested an amplicon-based method for resequencing the BRCA1 and BRCA2 genes on an Illumina MiSeq to identify disease-causing mutations in patients with hereditary breast or ovarian cancer (HBOC). The coding regions of BRCA1 and BRCA2 were resequenced in 96 HBOC patient DNA samples obtained from different sample types: peripheral blood leukocytes, whole blood drops dried on paper, and buccal wash epithelia. A total of 16 random DNA samples were characterized using standard Sanger sequencing and applied to optimize the variant calling process and evaluate the accuracy of the MPS-method. The best bioinformatics workflow included the filtration of variants using GATK with the following cut-offs: variant frequency >14%, coverage (>25x) and presence in both the forward and reverse reads. The MPS method had 100% sensitivity and 94.4% specificity. Similar accuracy levels were achieved for DNA obtained from the different sample types. The workflow presented herein requires low amounts of DNA samples (170 ng) and is cost-effective due to the elimination of DNA and PCR product normalization steps.
Subject(s)
BRCA1 Protein/genetics , BRCA2 Protein/genetics , Breast Neoplasms/genetics , High-Throughput Nucleotide Sequencing/methods , Ovarian Neoplasms/genetics , Sequence Analysis, DNA/methods , Base Sequence , DNA Mutational Analysis/methods , Female , Genetic Testing , HumansABSTRACT
In the present study, new variable number tandem repeats (VNTR) loci in the Neisseria gonorrhoeae genome were identified in silico. VNTR analysis scheme using PCR and agarose or polyacrylamide gel electrophoresis was developed based on nine VNTR loci with various degrees of polymorphism. The method was used to genotype a collection of 48 isolates, obtained from patients with gonorrhea in Almaty, Kazakhstan during the period from December 2008 to November 2009. This collection of isolates was also characterized by the opa-typing and multiantigen sequence typing (NG-MAST). The discriminatory power of the VNTR analysis translated by Hunter-Gaston Discrimination Index (HGDI) was similar to that of opa typing (HGDI=0.98 versus 0.97) and slightly higher than that of NG-MAST (HDGI=0.95). The adjusted Rand (AR) coefficients and Wallace coefficients showed that the overall concordance between the typing methods was not high. VNTR analysis described here is simple, inexpensive, easy to interpret, and it would be reliable for the comparison of data obtained in different laboratories. The proposed VNTR loci might be used for epidemiological studies of gonococcal infections.
