Phosphoramidate Azole Oligonucleotides for Single Nucleotide Polymorphism Detection by PCR.

Detection of the Kirsten rat sarcoma gene (KRAS) mutational status is an important factor for the treatment of various malignancies. The most common KRAS-activating mutations are caused by single-nucleotide mutations, which are usually determined by using PCR, using allele-specific DNA primers. Oligonucleotide primers with uncharged or partially charged internucleotide phosphate modification have proved their ability to increase the sensitivity and specificity of various single nucleotide mutation detection. To enhance the specificity of single nucleotide mutation detection, the novel oligonucleotides with four types of uncharged and partially charged internucleotide phosphates modification, phosphoramide benzoazole (PABA) oligonucleotides (PABAO), was used to prove the concept on the KRAS mutation model. The molecular effects of different types of site-specific PABA modification in a primer or a template on a synthesis of full-length elongation product and PCR efficiency were evaluated. The allele-specific PCR (AS-PCR) on plasmid templates showed a significant increase in analysis specificity without changes in Cq values compared with unmodified primer. PABA modification is a universal mismatch-like disturbance, which can be used for single nucleotide polymorphism discrimination for various applications. The molecular insights of the PABA site-specific modification in a primer and a template affect PCR, structural features of four types of PABAO in connection with AS-PCR results, and improvements of AS-PCR specificity support the further design of novel PCR platforms for various biological targets testing.

Molecular characterization of Mycoplasma pneumoniae infections in Moscow from 2015 to 2018.

The aim of this study was to assess which Mycoplasma pneumoniae genotypes were present in Moscow during the years 2015-2018 and whether the proportion between detected genotypes changed over time. We were also interested in the presence of macrolide resistance (MR)Mycoplasma pneumoniae. We performed multilocus variable-number tandem-repeat (VNTR) analysis (MLVA), SNP typing, and mutation typing in the 23S rRNA gene from 117 M. pneumoniae clinical isolates. Our analysis suggests two major MLVA types: 4572 and 3562. In 2017-2018, MLVA type 4572 gradually became predominant. In general, the SNP type range is the same as described earlier for European countries. The analysis of MR mutations showed that 7% of the isolates had an A2063G mutation in the 23S rRNA gene with no isolates carrying an A2064G mutation. In 2017-2018, MLVA type 4572 (SNP type 1) begins to spread in Moscow, which was widespread globally, especially in Asian countries. SNP typing of our sample showed higher discriminatory power than MLVA typing.

Angiopoietin-Like Proteins 4 (ANGPTL4) Gene Polymorphisms and Risk of Brain Arteriovenous Malformation.

BACKGROUND: Brain arteriovenous malformations (BAVMs) are formed by hypertrophied arterial vessels (afferents, feeders), a large number of arteriovenous shunts which become tangled to form a body (nidus) of malformation, which then expands draining proximal veins. The aim of this study was a replication of single nucleotide polymorphism (SNP) rs11672433 association with BAVM development with the subsequent meta-analysis of published data. METHODS: A total of 252 Russian patients with brain BAVMs and 480 control subjects were included in the present study. Genotyping was performed using real-time polymerase chain reaction with competitive hydrolysis probes. RESULTS: In our case-control study, we found no significant association with brain arteriovenous malformation for the SNP rs11672433 of ANGPTL4 gene (odds ratio .82, 95% confidence interval = .57-1.17 P value = .27) as well as in meta-analysis (odds ratio 1.18, 95% confidence interval = .81-1.73, P value = .39). CONCLUSIONS: Our data showed that SNP rs11672433 was not associated with the BAVM Russian population and the following meta-analysis did not detect an association in total. Thus, in spite of the fact that ANGPTL4 (protein) participates in the angiogenesis regulation processes, we consider that SNP rs11672433, a high-frequency locus in the ANGPTL4 gene, does not influence the predisposition to BAVM or its effect is too small to be detected in the present size sample set.

TERT polymorphisms rs2853669 and rs7726159 influence on prostate cancer risk in Russian population.

Telomere length and telomerase activity have been hypothesized to play a role in cancer development. The aim of our study was to investigate the association of allelic variants of three functional polymorphisms rs2853669, rs2736100, and rs7726159 in the telomerase reverse transcriptase (TERT) gene with the risk of the breast cancer and prostate cancer in Russian population. Six hundred sixty women with breast cancer, 372 men with prostate cancer, and corresponding control groups of 523 women and 363 men were included in the present case-control study. We observed an association of allele rs2853669 C with increased risk of prostate cancer (co-dominant model TC vs. TT OR = 1.65, P = 0.002; additive model OR = 1.42, P = 0.005; dominant model: OR = 1.64, P = 0.001) and allele rs7726159 A with reduced risk of this malignancy (сo-dominant model: AA vs. CC OR = 0.42, P = 0.002; additive model: OR = 0.69, P = 0.002; dominant model: OR = 0.67, P = 0.01; recessive model: OR = 0.48, P = 0.005). None of the studied polymorphisms showed an association with the risk of breast cancer. Our results provide evidence that the TERT gene variability modulate prostate cancer predisposition in ethnical Russians.

Characterization of extensively drug-resistant Mycobacterium tuberculosis isolates circulating in Siberia.

BACKGROUND: The spread of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis compromises effective control of tuberculosis (TB) in Siberia. Early identification of drug-resistant isolates is, therefore, crucial for effective treatment of this disease. The aim of this study was to conduct drug susceptibility testing and identify mutations in drug resistance genes in clinical isolates of M. tuberculosis from some TB patients presenting for treatment in Siberia. METHODS: Thirty randomly selected clinical isolates of M. tuberculosis were obtained from the Novosibirsk Research Institute of Tuberculosis, Russia. Isolates were screened for drug resistance and characterized by variable number of tandem repeats (VNTR)-typing using 15 standard and four additional loci. Deligotyping on multiple large sequences was performed using 10 loci. RESULTS: Twenty-nine of the isolates were assigned XDR status. Twenty-eight isolates belonged to the M. tuberculosis Beijing family, from which 11 isolates were considered the M11 type (39%), two the M2 type (7%), and one the M33 type (3%). Seventeen isolates (60.7%) from this family exhibited unique genetic patterns. The remaining two isolates belonged to the Latino-American Mediterranean family. Gene sequences (rpoB, katG, rrs, rpsL, tlyA, gidB, gyrA, gyrB) were analyzed to identify mutations that confer resistance to rifampicin, isoniazid, amikacin, kanamycin, capreomycin, and ofloxacin. The most common mutations among the XDR isolates were S531L in RpoB, S315T in KatG, various codon 94 mutations in gyrA, A90V in GyrA, K43R in RpsL, and 1401 A → G in rrs; these confer resistance to rifampicin, isoniazid, ofloxacin, streptomycin and kanamycin/capreomycin, respectively. There was high congruence between the two typing methods (VNTR typing and deligotyping) and RD105, RD149, RD152, RD181, and RD207 regions of difference were absent from the 28 Beijing family isolates. CONCLUSIONS: Deligotyping can be used for rapid and reliable screening of M. tuberculosis isolates, followed by more in-depth genotyping. Identification of Beijing family isolates with extensive drug resistance confirms that such strains have epidemiological importance in Siberia. Rapid detection of mutations that lead to drug resistance should facilitate selection of effective drug therapies, and the development of early prevention strategies to combat this infection.

Loci cg06256735 and cg15815843 in the MFAP5 gene regulatory regions are hypomethylated in varicose veins apparently due to active demethylation.

Varicose vein disease (VVD) is a common health problem worldwide. Microfibril-associated protein 5 (MFAP5) is one of the potential key players in its pathogenesis. Our previous microarray analysis revealed the cg06256735 and cg15815843 loci in the regulatory regions of the MFAP5 gene as hypomethylated in varicose veins which correlated with its up-regulation. The aim of this work was to validate preliminary microarray data, estimate the level of 5-hydroxymethylcytosine (5hmC) at these loci, and determine the methylation status of one of them in different layers of the venous wall. For this, methyl- and hydroxymethyl-sensitive restriction techniques were used followed by real-time PCR and droplet digital PCR, correspondingly, as well as bisulfite pyrosequencing of +/- oxidized DNA. Our microarray data on hypomethylation at the cg06256735 and cg15815843 loci in whole varicose vein segments were confirmed and it was also demonstrated that the level of 5hmC at these loci is increased in VVD. Specifically, among other layers of the venous wall, tunica (t.) intima is the main contributor to hypomethylation at the cg06256735 locus in varicose veins. Thus, it was shown that hypomethylation at the cg06256735 and cg15815843 loci takes place in VVD, with evidence to suggest that it happens through their active demethylation leading to up-regulation of the MFAP5 gene, and t. intima is most involved in this biochemical process.

Association GSTT1, GSTM1 and GSTP1 (Ile105Val) genetic polymorphisms in mothers with risk of congenital malformations in their children in Western Siberia: a case-control study.

OBJECTIVE: Polymorphisms of glutathione S-transferase (GST) genes in mothers may be involved in teratogenesis in their offspring. This study aims to investigate the association of GST genes (T1, M1 and P1) with the risk of having children with congenital malformations (CMs) in residents of the West Siberian region of Russia. METHOD: We studied 235 women with offspring's with CMs, and 273 women with one or more healthy children. Null genotypes of GSTM1 and GSTT1 were identified through multiplex real-time polymerase chain reaction, and GSTP1 gene (Ile105Val) polymorphism was determined through TaqMan-real-time polymerase chain reaction. RESULTS: The study showed that the maternal genotype GSTT1 «0/0¼ is associated with CMs in the offspring (odd ratio (OR) = 3.63, P = 5.18 × 10(-9) ). A significant association of the maternal genotype GSTT1 «0/0¼ with CMs of the cardiovascular system (OR = 5.03, P = 2.93 × 10(-7) ), urinary system (OR = 4.20, P = 3.51 × 10(-6) ) and central nervous system (OR = 4.40, P = 6.69 × 10(-5) ) was found in the child. No association of maternal GSTM1 (del) and GSTP1 (Ile105Val) genetic polymorphisms with CMs of the child was identified. CONCLUSION: Homozygous deletion of the GSTT1 gene in women of the West Siberian region is a risk factor for birth defects in the child.

A Novel Thermostable and Processive Reverse Transcriptase from a Group II Intron of Anoxybacillus flavithermus.

Reverse transcriptases (RTs) are a family of enzymes that synthesize DNA using an RNA template and are involved in retrovirus propagation and telomere lengthening. In vitro, RTs are widely applied in various methods, including RNA-seq, RT-PCR, and RT-LAMP. Thermostable RTs from bacterial group II introns are promising tools for biotechnology due to their higher thermostability, fidelity, and processivity compared to commonly used M-MuLV RT and its mutants. However, the diversity of group II intron-encoded RTs is still understudied. In this work, we biochemically characterized a novel RT from a thermophilic bacterium, Anoxybacillus flavithermus, which was isolated from a hot spring in New Zealand and has an optimal growth temperature of around 60 °C. The cloned RT, named Afl RT, retained approximately 40% of the specific activity after a 45 min incubation at 50 °C. The optimal pH was 8.5, the optimal temperature was between 45 and 50 °C, and Mn2+ ions were found to be an optimal cofactor. The processivity analysis with MS2 phage gRNA (3569 b) demonstrated that Afl RT elongated fully up to 36% of the template molecules. In reverse transcription and RT-qLAMP, the enzyme allowed up to 10 copies of MS2 phage genomic RNA to be detected per reaction. Thus, Afl RT holds great potential for a variety of practical applications that require the use of thermostable and processive RTs.

Epigenome-Wide Changes in the Cell Layers of the Vein Wall When Exposing the Venous Endothelium to Oscillatory Shear Stress.

Epigenomic changes in the venous cells exerted by oscillatory shear stress towards the endothelium may result in consolidation of gene expression alterations upon vein wall remodeling during varicose transformation. We aimed to reveal such epigenome-wide methylation changes. Primary culture cells were obtained from non-varicose vein segments left after surgery of 3 patients by growing the cells in selective media after magnetic immunosorting. Endothelial cells were either exposed to oscillatory shear stress or left at the static condition. Then, other cell types were treated with preconditioned media from the adjacent layer's cells. DNA isolated from the harvested cells was subjected to epigenome-wide study using Illumina microarrays followed by data analysis with GenomeStudio (Illumina), Excel (Microsoft), and Genome Enhancer (geneXplain) software packages. Differential (hypo-/hyper-) methylation was revealed for each cell layer's DNA. The most targetable master regulators controlling the activity of certain transcription factors regulating the genes near the differentially methylated sites appeared to be the following: (1) HGS, PDGFB, and AR for endothelial cells; (2) HGS, CDH2, SPRY2, SMAD2, ZFYVE9, and P2RY1 for smooth muscle cells; and (3) WWOX, F8, IGF2R, NFKB1, RELA, SOCS1, and FXN for fibroblasts. Some of the identified master regulators may serve as promising druggable targets for treating varicose veins in the future.

Allele-Specific PCR for PIK3CA Mutation Detection Using Phosphoryl Guanidine Modified Primers.

Phosphoryl guanidine (PG) is the novel uncharged modification of internucleotide phosphates of oligonucleotides. Incorporating PG modification into PCR primers leads to increased discrimination between wild-type and mutated DNA, providing extraordinary detection limits in an allele-specific real-time polymerase chain reaction (AS-PCR). Herein, we used PG-modification to improve the specificity of AS primers with unfavorable Pyr/Pur primer's 3'-end mismatch in the template/primer complex. Two mutations of the PIK3CA gene (E542K, E545K) were chosen to validate the advantages of the PG modification. Several primers with PG modifications were synthesized for each mutation and assessed using AS-PCR with the plasmid controls and DNA obtained from formalin-fixed paraffin-embedded (FFPE) tissues. The assay allows the detection of 0.5% of mutated DNA on the wild-type DNA plasmid template's background with good specificity. Compared with ddPCR, the primers with PG-modification demonstrated 100% specificity and 100% sensitivity on the DNA from FFPE with mutation presence higher than 0.5%. Our results indicate the high potential of PG-modified primers for point mutation detection. The main principle of the developed methodology can be used to improve the specificity of primers regardless of sequences.

Polymorphisms in folate-metabolizing genes and risk of having an offspring with congenital anomalies in the West Siberian region of Russia: a case-control study.

OBJECTIVE: Periconceptional folate supplementation prevents a number of congenital anomalies (CA). The aim of our study was to investigate the association of 11 polymorphisms in the folate-metabolizing genes with the risk of having an offspring with CA in the Russian ethnic group. METHOD: We genotyped 280 mothers having a CA-affected pregnancy and 390 control mothers. The most common malformations among the cases were CA of the nervous, urinary, and cardiovascular systems, and these groups were analyzed separately. RESULTS: In the whole group of CA, we revealed the associations of MTHFR C677T and MTR A2756G loci with increased risk of CA-affected pregnancy. In the group of CA of the cardiovascular system, we observed an association of MTHFR A1298C with decreased risk and an association of MTR A2756G with increased risk of CA. After the Bonferroni correction, only the association between the genotype MTR 2756GG and the risk of having a fetus with CA of the cardiovascular system remained statistically significant (OR = 4.99, P = 0.03). CONCLUSION: These findings indicate that locus A2756G in the MTR gene may play a role in susceptibility to CA of the cardiovascular system in West Siberia, but further research is necessary to confirm the association.

Comparison of Reverse Transcriptase (RT) Activities of Various M-MuLV RTs for RT-LAMP Assays.

Reverse transcriptases (RTs) are a family of enzymes synthesizing DNA using RNA as a template and serving as indispensable tools in studies related to RNA. M-MuLV RT and its analogs are the most commonly used RTs. RTs are widely applied in various diagnostics methods, including reverse-transcription loop-mediated isothermal amplification (RT-LAMP). However, the performance of different RTs in LAMP remains relatively unknown. Here, we report on the first direct comparison of various M-MuLV RTs in RT-LAMP, including enzymes with a different number of mutations and fusions with Sto7d. Several parameters were assessed, namely: optimal reaction temperature, enzyme concentration, reverse transcription time, a minimal amount of RNA template, and tolerance to inhibitors. Mutations increased the optimal reaction temperature from 55 °C to 60-65 °C. All of the RTs were suitable for RT-LAMP with RNA templates in the range of 101-106 copies per reaction. Highly mutated enzymes were 1.5-3-fold more tolerant to whole blood, blood plasma, and guanidinium, but they were two-fold more sensitive to high concentrations of NaCl. The comparison of different RTs presented here could be helpful for selecting the optimal enzyme when developing novel LAMP-based diagnostic tests.

Development of a multiplex allele-specific qPCR approach for testing PIK3CA mutations in patients with colorectal cancer.

Phosphatidylinositol 3-kinases (PI3Ks) are lipid kinases involved in cellular growth and division. Somatic mutations in one of the PI3K catalytic subunit genes, PIK3CA, are frequently found in numerous malignancies, including colorectal cancer (CRC). Several PIK3CA inhibitors are approved for the treatment of breast cancer and lymphoma. Activating mutations in PIK3CA tend to occur in exons 9 and 20, with mutations in other exons 1, 4, and 7 being less common. Most test systems for PIK3CA mutation screening are designed to detect mutations in exons 9 and 20, leaving exons 1-7 overlooked. We have developed a multiplex AS-PCR to screen for PIK3CA mutations in exons 1, 4, 7, 9, and 20. Validation was performed on 515 CRC samples of patients from Siberia and the Far East of Russia. The assay sensitivity was 0.05-0.5% of mutant DNA, and the overall PIK3CA mutation frequency was 13.01%, with 9.32% of mutations in exon 9, 1.94% in exon 20, and 1.74% in exons 1-7. The assay designed is suitable for the analysis of activating PIK3CA mutations in formalin-fixed paraffin-embedded tissue samples. The present work is the first study characterizing the PIK3CA mutation frequency in CRC patients from the eastern part of Russia.

Quantitative and structural characteristics of mitochondrial DNA in varicose veins.

OBJECTIVE: We examined quantitative (in terms of mtDNA/nuclear DNA) and structural (in terms of common deletions in the MT-ND4 gene region) characteristics of mitochondrial DNA (mtDNA) in varicose veins (VVs) and venous wall layers by comparing mitochondrial genome parameters, as well as mitochondrial function (in terms of mitochondrial membrane potential (MtMP)), in varicose vein (VV) vs. non-varicose vein (NV) tissue samples. METHODS: We analyzed paired great saphenous vein samples (VV vs. NV segments from each patient left after venous surgery) harvested from patients with VVs. Relative mtDNA level and the proportion of no-deletion mtDNA were determined by a multiplex quantitative PCR (qPCR), confirming the latter with a more sensitive method - droplet digital PCR (ddPCR). Mitochondria's functional state in VVs was assessed using fluorescent (dependent on MtMP) live-staining of mitochondria in venous tissues. RESULTS: Total mtDNA level was lower in VV than in NV samples (predominantly in the t. media layer). ddPCR analysis showed lower proportion of no-deletion mtDNA in VVs. Because of the decrease in relative MtMP in VVs, our results suggest a possible reduction of mitochondrial function in VVs. CONCLUSION: Quantitative and structural changes (copy number and integrity) of mtDNA are plausibly involved in VV pathogenesis. Future clinical studies implementing the mitochondrial targeting may be eventually fostered after auxiliary mechanistic studies.

Multiplex Droplet Digital PCR Assay for Detection of MET and HER2 Genes Amplification in Non-Small Cell Lung Cancer.

Non-small-cell lung cancer (NSCLC), a subtype of lung cancer, remains one of the most common tumors with a high mortality and morbidity rate. Numerous targeted drugs were implemented or are now developed for the treatment of NSCLC. Two genes, HER2 and MET, are among targets for these specific therapeutic agents. Alterations in HER2 and MET could lead to primary or acquired resistance to commonly used anti-EGFR drugs. Using current methods for detecting HER2 and MET amplifications is time and labor-consuming; alternative methods are required for HER2 and MET testing. We developed the first multiplex droplet digital PCR assay for the simultaneous detection of MET and HER2 amplification in NSCLC samples. The suitability of qPCR was assessed for the optimization of multiplex ddPCR. The optimal elongation temperature, reference genes for DNA quantification, and amplicon length were selected. The developed ddPCR was validated on control samples with various DNA concentrations and ratios of MET and HER2 genes. Using ddPCR, 436 EGFR-negative NSCLC samples were analyzed. Among the tested samples, five specimens (1.15%) showed a higher ratio of MET, and six samples (1.38%) showed a higher ratio of HER2. The reported multiplex ddPCR assay could be used for the routine screening of MET and HER2 amplification in NSCLC samples.

Polymorphic locus rs10492972 of the KIF1B gene association with multiple sclerosis in Russia: case control study.

Axonal degeneration is responsible for the progression of the irreversible destruction caused by multiple sclerosis (MS) resulting ultimately in permanent disability. The KIF1B protein, a member of the kinesin family, is necessary for axon growth and myelination in vertebrates. In the recent paper, Aulchenko et al. suggested that the rs10492972[C] variant of KIF1B increases susceptibility to MS, but three following replication study didn't confirm this association. We studied the association of the polymorphic locus rs10492972 present in the KIF1B gene with genetic predisposition and its occurrence in clinical presentations of MS patients resident in western Siberia and the Sakha Republic (Yakutia), Russia. rs10492972 has been genotype in 833 samples of MS patient and 689 healthy controls. Distribution of rs10492972 genotypes corresponded with a Hardy-Weinberg distribution in both the MS patient and control groups, with the frequency of the C allele being the same in both groups (33%). Frequencies of occurrence of the genotypes were not shown to be associated with different disease courses or other characteristics of the disease, such as age at onset or duration. A complete meta-analysis of all analogous studies published to date showed that the protective effect of the rs10492972[C] allele is statistically significant (OR=0.95, C.I.95% [0.90-0.99], p=0.02).

Genetic variation of Mycobacterium tuberculosis circulating in Kharkiv Oblast, Ukraine.

BACKGROUND: A persistent increase of tuberculosis cases has recently been noted in the Ukraine. The reported incidence of drug-resistant isolates of M. tuberculosis is growing steadily; however, data on the genetic variation of isolates of M. tuberculosis circulating in northern Ukraine and on the spectrum and frequency of occurrence of mutations determining resistance to the principal anti-tuberculosis drugs isoniazid and rifampicin have not yet been reported. METHODS: Isolates of M. tuberculosis from 98 tuberculosis patients living in Kharkiv Oblast (Ukraine) were analyzed using VNTR- and RFLP-IS6110-typing methods. Mutations associated with resistance to rifampicin and isoniazid were detected by RFLP-PCR methods, and also confirmed by sequencing. RESULTS: We identified 75 different genetic profiles. Thirty four (34%) isolates belonged to the Beijing genotype and 23 (23%) isolates belonged to the LAM family. A cluster of isolates belonging to the LAM family had significant genetic heterogeneity, indicating that this family had an ancient distribution and circulation in this geographical region. Moreover, we found a significant percentage of the isolates (36%) belonged to as yet unidentified families of M. tuberculosis or had individual non-clustering genotypes. Mutations conferring rifampicin and isoniazid resistance were detected in 49% and 54% isolates, respectively. Mutations in codon 531 of the rpoB gene and codon 315 of the katG gene were predominant among drug-resistant isolates. An association was found for belonging to the LAM strain family and having multiple drug resistance (R = 0.27, p = 0.0059) and also for the presence of a mutation in codon 531 of the rpoB gene and belonging to the Beijing strain family (R = 0.2, p = 0.04). CONCLUSIONS: Transmission of drug-resistant isolates seems to contribute to the spread of resistant TB in this oblast. The Beijing genotype and LAM genotype should be seen as a major cause of drug resistant TB in this region.

M-MuLV reverse transcriptase: Selected properties and improved mutants.

Reverse transcriptases (RTs) are enzymes synthesizing DNA using RNA as the template and serving as the standard tools in modern biotechnology and molecular diagnostics. To date, the most commonly used reverse transcriptase is the enzyme from Moloney murine leukemia virus, M-MuLV RT. Since its discovery, M-MuLV RT has become indispensable for modern RNA studies; the range of M-MuLV RT applications is vast, from scientific tasks to clinical testing of human pathogens. This review will give a brief description of the structure, thermal stability, processivity, and fidelity, focusing on improving M-MuLV RT for practical usage.

Allele-Specific PCR for KRAS Mutation Detection Using Phosphoryl Guanidine Modified Primers.

Establishing the Kirsten rat sarcoma (KRAS) mutational status is essential in terms of managing patients with various types of cancer. Allele-specific real-time polymerase chain reaction (AS-PCR) is a widely used method for somatic mutations detection. To improve the limited sensitivity and specificity, several blocking methods have been introduced in AS-PCR to block the amplification of wild-type templates. Herein, we used a novel modified oligonucleotide with internucleotide phosphates reshaped 1,3-dimethyl-2-imino-imidazolidine moieties (phosphoryl guanidine (PG) groups) as primers and blockers in the AS-PCR method. Four common KRAS mutations were chosen as a model to demonstrate the advantages of the PG primers and blockers utilizing a customized PCR protocol. The methods were evaluated on plasmid model systems providing a KRAS mutation detection limit of 20 copies of mutant DNA in a proportion as low as 0.1% of the total DNA, with excellent specificity. PG-modification can serve as the universal additional mismatch-like disturbance to increase the discrimination between wild-type and mutated DNA. Moreover, PG can serve to increase primer specificity by a synergetic effect with additional mismatch and would greatly facilitate medical research.

Absence of EGFR C797S Mutation in Tyrosine Kinase Inhibitor-Naïve Non-Small Cell Lung Cancer Tissues.

EGFR tyrosine-kinase inhibitors (TKIs) are used as targeted therapeutics for the treatment of advanced non-small cell lung cancer (NSCLC) with EGFR-activating mutations. EGFR C797S is common causes of acquired resistance to third-generation TKIs. There is wide-spread opinion that resistance-conferring mutation present even in a small proportion of cancer cells before the start of therapy could potentially predict poor response to a targeted drug. In our study, we tested whether C797S can be found in previously untreated NSCLCs. We analyzed DNA samples extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissue sections of 470 lung adenocarcinoma patients, including 235 samples with activating EGFR mutations. Screening was performed using highly sensitive droplet digital PCR assay. No tumor samples with baseline C797S were identified. C797S does not occur in TKI-naïve NSCLCs and provide evidence that screening for this mutation before TKIs administration may not be necessary.