Allele-specific Primers Research Articles

Improvement of HER2I655V TARMS-PCR Performance by DNA Quality Analysis

Reliable TARMS-PCR is a prerequisite in constructing a solid conclusion in genetic diagnostics. The validity of data generated by this molecular technique is hampered by a false positive result. In attempt to develop a TARMS-PCR for HER2I655V genotyping with no interfering of bias we used DNase I to eliminate DNA contaminant resided in PCR reagent. TARMS-PCR without enzyme treatment using recombinant plasmids that contained HER2I655V gene with represented its alleles was used to evaluate the presence of false positive result while DNase I treated-PCR reagent was used in TARMS-PCR to evaluate the effective dose of the enzyme and further to adjust the TARMS-PCR conditions. PCR master mix kit used in this study produced a false positive result on HER2I655V TARMS-PCR as proven by the presence of multiple PCR products in Non-Template Control (NTC) and 0.1 U of the enzyme could eliminate this DNA contaminant effectively, although this pretreatment altered the specificity of HER2I655V TARMS-PCR genotyping on certain genotype. Combination of touchdown TARMS-PCR with another allele-specific primer recovered specificity of detection on this model system. Interestingly, this optimized HER2I655V TARMS-PCR can only be used for genotyping the clinical samples if only further optimization was done using genomic DNA as template

Large enthesophytes in teenage skulls: Mechanical, inflammatory and genetic considerations

BackgroundThe literature implies that large enthesophytes are exclusive to genetically predisposed individuals and to Spondyloarthropathies sufferers. Accordingly, the aim of this investigation and report was to assess the involvement of genetic predisposition, inflammatory and/or mechanical influences in the development of large enthesophytes in a sample population of teenagers presenting with large enthesophytes emanating from the external occipital protuberance. MethodsAnalysis was based on four teenage males (13–16 year-old) possessing 14.5–30.5 mm enthesophytes projecting from the external occipital protuberance. This study included assessment of radiographs, MRI scans, blood-work, history, the SF-36 health survey, and the comparison of these data with the relevant literature to describe the interrelationships between the presence of enlarged external occipital protuberance, forward head protraction, active inflammation and/or genetic factors. FindingsKnown genetic markers (e.g. HLA-B27) were not detected by allele-specific primers and both ESR and CRP tests were negative. Additionally, MRI analyses failed to detect active localised inflammation at the external occipital protuberance and surrounding structures. The health survey yielded normal parameters for all participants. All participants displayed significantly large Forward Head Protraction values (>40 mm), and interviews with participants and their parents indicated that concerns related to posture were prevalent since early childhood. InterpretationThis report suggests that mechanical load has an important role in enthesophyte development, irrespective the involvement of inflammatory or genetic factors.

The reversion of A23525T of polymorphism of FTO gene in persons with overweight in conditions of hypocaloric diet

ObjectiveTo study the association of A23525T polymorphism of the FTO gene with overweight/obesity and the possibility of reversion of single-nucleotide replacements rs9939609 in a hypocaloric diet. Materials and methodsPolymorphisms of the FTO gene (A23525T, rs9939609) are typified by the SNP method (single nucleotide polymorphism) with allele-specific primers on the test systems of the NPF "Liteh".The frequencies rs9939609 were studied in persons with overweight, I–III degree of obesity (n = 84) compared with the control group (n = 32). The reversion of polymorphic variants of genes under the influence of a hypocaloric diet has been analyzed. The experimental data are processed in SPSS Statistics 22.0. ResultsIn the pilot study, unlike previously published studies, a reliable association of the genotype of the FTO gene with a violation of lipid metabolism (OR = 2.33, CI 95%) was shown not by A23525A, but by T23525T. A positive correlation was established between the mean force between the TT genotype and the weight of the participants in the experiment (r = + 0.625, p = 0.022), fat mass (r = + 0.610; p = 0.046), body mass index (BMI) (r = + 0.577; p = 0.039) and the circumference of the hips (r = + 0.636; p = 0.039). In 25 out of 84 participants (29.7%) after RTD, a partial or complete reversal (reversal) of nucleotides occurred in rs9939609: 40% of them were mutations transversion caused by the replacement of T→A nucleotides and the A23525T, T23525T transition of genotypes into the homozygous A23525A variant. ConclusionThe TT genotype of the FTO gene is associated with obesity. The reversal of polymorphisms in the FTO gene under the influence of RDT is mainly associated with transversion of nucleotides (А→Т) at the corresponding site (23525) of the intron mutation (rs9939609).

Donor Genotype in the Interleukin-7 Receptor α-Chain Predicts Risk of Graft-versus-Host Disease and Cytomegalovirus Infection after Allogeneic Hematopoietic Stem Cell Transplantation.

The efficacy of allogeneic hematopoietic stem cell transplantation (HSCT) is challenged by acute and chronic graft-versus-host disease (aGVHD and cGVHD) and viral infections due to long-lasting immunodeficiency. Interleukin-7 (IL-7) is a cytokine essential for de novo T cell generation in thymus and peripheral T cell homeostasis. In this study, we investigated the impact of the single nucleotide polymorphism rs6897932 in the IL-7 receptor α-chain (IL-7Rα) which has previously been associated with several autoimmune diseases. We included 460 patients undergoing allogeneic HSCT after a myeloablative conditioning. Patients had a median age of 26.3 years (0.3–67.0 years), and 372 (80.9%) underwent HSCT for malignant diseases. Donors were matched sibling donors (n = 147), matched unrelated donors (n = 244) or mismatched unrelated donors (n = 69), and the stem cell source were either bone marrow (n = 329) or peripheral blood (n = 131). DNA from donors was genotyped for the IL-7Rα single nucleotide polymorphism (SNP) rs6897932 using an allele-specific primer extension assay (CC: n = 252, CT: n = 178, TT: n = 30). The donor T allele was associated with a higher risk of grades III–IV aGVHD (HR = 2.0, 95% CI = 1.1–3.8, P = 0.034) and with significantly increased risk of extensive cGVHD (HR = 2.0, 95% CI = 1.1–3.6, P = 0.025) after adjustment for potential risk factors. In addition, the TT genotype was associated with a higher risk of cytomegalovirus (CMV) infection post-transplant (HR = 2.4, 95% CI = 1.2–4.3, P = 0.0068). Numbers of T cells were significantly higher on day +60 in patients receiving a rs6897932 TT graft (CD3+: 109% increase, P = 0.0096; CD4+: 64% increase, P = 0.038; CD8+: 133% increase, P = 0.011). Donor heterozygosity for the T allele was associated with inferior overall survival (HR = 1.7, 95% CI = 1.2–2.3, P = 0.0027) and increased treatment-related mortality (HR = 2.3, 95% CI = 1.3–4.0, P = 0.0047), but was not associated with the risk of relapse (P = 0.35). In conclusion, the IL-7Rα rs6897932 genotype of the donor is predictive of aGVHD and cGVHD, CMV infection, and mortality following HSCT. These findings indicate that IL-7Rα SNP typing of donors may optimize donor selection and facilitate individualization of treatment in order to limit treatment-related complications.

Role of gene polymorphism of the renin-angiotensin system components in development of cardiovascular diseases, excess body weight and obesity in inhabitants of the Adyghea Republic

The aim of the study was to study the polymorphisms of the AGT (Met235Thr; rs699), ACE (Ins/Del; rs4646994) and AGTR1 (A1166C; rs5186) genes associated with both CVD risk and obesity in the Republic of Adygea. Materials and methods. Polymorphisms (rs699, rs4646994,rs5186) are investigated by the single nucleotide polymorphism method (SNP) using allelespecific primers in the SNP-express test systems of NPF Liteh with electrophoretic detection of results. Distribution of frequencies and associations of AGT, ACE, AGTR1 gene mutations has been analyzed in control group (donors, n = 143) of young men and girls at the age of 22.32 ± 5.4 years without hereditary burden and clinical manifestations of cardiovascular disease, as well as at patients (n=46; 54.26 ± 12.2 years) of the Cardiological Department of the Adyghe Republican Clinical Hospital. Experimental data have been analysed by statistical methods using the SPSS Statistics 17.0 program. Results. A group of patients with the verified diagnoses of heart continuum diseases (DWC), show a statistically significantly increasedn frequency of mutant 1166C locus (p = 0.00003; OR = 3.77 (2.22–6.38)) and pathological homozygous C1166C genotype (р = 0.00002; OR = 10.36 (4.69–26.22)) of AGTR1 gene. No reliable distinctions have been revealed in a distribution of Met235Thr (AGT), and Ins/ Del (ACE) alleles in the examined groups of donors and patients at inhabitants of Adyghea Republic, as well as no correlation relationships have been identified between frequencies of Met235Thr, Ins/Del, and A1166C polymorphisms and obesity. Minor 1166C polymorphism of the AGTR1 gene is associated with heart continuum diseases and can be used as an informative predictor of cardiovascular diseases for the formation of risk groups, especially among persons of young age. The absence of associations between Met235Thr, Ins/Del and A1166C allelic variants of genes and dyslipidemia points to need of the analysis of some other AGT, ACE and AGTR1 gene polymorphisms obtained as a result of research in the Adyghea Republic.

Extended Donor Typing by Pooled Capillary Electrophoresis: Impact in a Routine Setting

Background: PCR with sequence-specific priming using allele-specific fluorescent primers and analysis on a capillary sequencer is a standard technique for DNA typing. We aimed to adapt this method for donor typing in a medium-throughput setting. Methods: Using the Extract-N-Amp PCR system, we devised a set of eight multiplex allele-specific PCR with fluorescent primers for Fy^a/Fy^b, Jk^a/Jk^b, M/N, and S/s. The alleles of a gene were discriminated by the fluorescent color; donor and polymorphism investigated were encoded by product length. Time, cost, and routine performance were collated. Discrepant samples were investigated by sequencing. The association of new alleles with the phenotype was evaluated by a step-wise statistical analysis. Results: On validation using 312 samples, for 1.1% of antigens (in 5.4% of samples) no prediction was obtained. 99.96% of predictions were correct. Consumable cost per donor were below EUR 2.00. In routine use, 92.2% of samples were successfully predicted. Discrepancies were most frequently due to technical reasons. A total of 11 previously unknown alleles were detected in the Kell, Lutheran, and Colton blood group systems. In 2017, more than 20% of the RBC units prepared by our institution were from donors with predicted antigen status. A steady supply of Yt(a-), Co(a-) and Lu(b-) RBC units was ensured. Conclusions: Pooled capillary electrophoresis offers a suitable alternative to other methods for extended donor DNA typing. Establishing a large cohort of typed donors improved transfusion support for patients.

Detection of G143A mutation in Erysiphe necator and its implications for powdery mildew management in grapes

Quinone outside inhibitor (QoI) fungicides are used worldwide for the management of Erysiphe necator but with associated problem of resistance development in the pathogen. Twenty nine E. necator isolates were collected during 2015–2016 from different geographical regions of India. In leaf disc bioassay using azoxystrobin, the EC50 of four isolates from research farms was <1 μg/ml, while 25 isolates from commercial vineyards had EC50 more than 115 μg/ml. The 256 fold resistance factor indicated G143A mutation. All the resistant isolates produced a 100 bp PCR product with G143A mutant allele specific primer which was not produced by the four sensitive isolates. A primer pair was designed for partial amplification of cytochrome b gene (Cyt b) and used for amplification of the gene from two resistant and two sensitive isolates. Alignment of amino acid sequences showed that the QoI resistant isolates harboured a G143A mutation, which was absent in the sensitive isolates. The two haplotypes of Cyt b gene from a resistant isolate, SAA2, and a sensitive isolate, HP1, have been deposited in GenBank under accession numbers KY418049 and KY418048, respectively. This is the first report of presence of QoI resistant isolates of E. necator from India. Studies point out the need for developing resistance management strategies by interspersing bio-control agents with judicious use of fungicides.

Allele-Specific Detection of SLC22A2 rs316019 Variants Associated with Metformin Disposition through the Kidney

Background: Metformin is prescribed as a first-line drug to treat type 2 diabetes. It is excreted directly and primarily through the SLC22A2 gene-encoded OCT2 transporter in the kidney. rs316019 (c.808G>T, p.270A>S) is the most common variant of SLC22A2, which affects its capacity to clear metformin from the body. Metformin increases the plasma lactate level in a concentration-dependent manner by inhibiting mitochondrial respiration and may lead to a condition known as metformin-associated lactic acidosis (MALA). MALA is a potentially life-threatening complication that can occur within the clinical doses of metformin. Therefore, dose adjustments based on the SLC22A2 rs316019 variants may be beneficial to maximize the efficacy and minimize the toxicity of metformin. Objective: This study was carried out to develop a simple and fast method to define genotype at the rs316019 locus. This method was applied to estimate the rs316019 allele frequencies in the Bangladeshi population. Methods: We designed allele-specific primers to determine genotype at the rs316019 locus using allele-specific polymerase chain reaction (AS-PCR). AS-PCR data were confirmed by targeted sequencing of randomly selected samples. Results: The DNA sequence chromatograms showed the exact genotypes predicted through the AS-PCR method. A proportion of 79.62, 18.01, and 2.37% of Bangladeshi individuals have GG, GT, and TT genotypes, respectively. Conclusion: We report here a simple and fast method to define ge­notypes at the rs316019 locus in diabetic patients who are under metformin regimen. Allele frequencies at the rs316019 locus in the Bangladeshi population are close to those reported in other populations.

Improvement of HER2I655V TARMS-PCR Performance by DNA Quality Analysis

Reliable TARMS-PCR is a prerequisite in constructing a solid conclusion in genetic diagnostics. The validity of data generated by this molecular technique is hampered by a false positive result. In attempt to develop a TARMS-PCR for HER2I655V genotyping with no interfering of bias we used DNase I to eliminate DNA contaminant resided in PCR reagent. TARMS-PCR without enzyme treatment using recombinant plasmids that contained HER2I655V gene with represented its alleles was used to evaluate the presence of false positive result while DNase I treated-PCR reagent was used in TARMS-PCR to evaluate the effective dose of the enzyme and further to adjust the TARMS-PCR conditions. PCR master mix kit used in this study produced a false positive result on HER2I655V TARMS-PCR as proven by the presence of multiple PCR products in Non-Template Control (NTC) and 0.1 U of the enzyme could eliminate this DNA contaminant effectively, although this pretreatment altered the specificity of HER2I655V TARMS-PCR genotyping on certain genotype. Combination of touchdown TARMS-PCR with another allele-specific primer recovered specificity of detection on this model system. Interestingly, this optimized HER2I655V TARMS-PCR can only be used for genotyping the clinical samples if only further optimization was done using genomic DNA as template

Marker assisted backcrossing for introgression of Fusarium wilt resistance gene into melon

Fusarium wilt, caused by Fusarium oxysporum f. sp. melonis is a common vascular wilt fungal disease in melon across the world. The resistance gene to race 1 of this causal agent, Fom-2, has been previously cloned and its sequence is available. The objective of this research was the introgression of Fom-2 from one resistant (Isabelle) genotype into two susceptible cultivars (Garmak and Tile-torogh) via marker assisted backcrossing. First, the leucine-rich repeats (LRR) domain of Fom-2 from resistant and susceptible genotypes was sequenced to develop functional markers. A length of 1274 bp of the 3′ end of this gene was isolated, cloned and sequenced. The difference between resistant and susceptible genotypes in this region was 28 nucleotide substitutions. Two allele specific primer pairs, Fom2-R409 and Fom2-S253, were designed based on nucleotide substitutions to amplify resistant and susceptible alleles, respectively. For introgression of the gene, donor (Isabelle) and recurrent (Garmak and Tile-torogh) parents were crossed. Resistant plants in BC1F1 and BC2F1 generations were first detected using artificial pathogen inoculation and later the plants were genotyped by functional markers to validate their resistance. The resistant plants were also selected phenotypically in each generation for background genome recovery, which conduced to high similarity of BC3 generation with the recurrent parents. It was proved the developed markers are more precise and efficient than inoculation trial and could be used as confident tools for screening of resistant melon genotypes to Fusarium wilt.

A Fluorescence Quenching Assay Based on Molecular Beacon Formation through a Ligase Detection Reaction for Facile and Rapid Detection of Point Mutations.

A fluorescence quenching assay based on a ligase detection reaction was developed for facile and rapid detection of point mutations present in a mixed population of non-variant DNA. If the test DNA carried a targeted mutation, then the two allele-specific primers were ligated to form a molecular beacon resulting in the expected fluorescence quenching signatures. Using this method, we successfully detected as low as 5% mutant DNA in a mixture of wild-type DNA (t test at 99% confidence level).

L55M and Q192R polymorphism of Paraoxonase gene and the risk of myocardial infarction in South Indian Tamil population

Paraoxonase, also known as aromatic esterase 1 encoded by PON1 gene is a high density lipoprotein linked enzyme that possess antiatherogenic activity. It protects low density lipoprotein (LDL) from oxidation and destroys biologically active oxidized lipids on lipoproteins and in arterial cells. Polymorphisms in PON1 gene, L55M and Q192R on exon 3 and 6 respectively are known to be risk in causing cardiovascular diseases. The study was aimed to determine the association of L55M and Q192R in pathogenicity of myocardial infarction in South Indian Tamil population. Tetra primer Amplification Refractory Mutation System–PCR method uses allele specific primer to discriminate the L55M and Q192R alleles. Total of 350 individuals enrolled at SRM Medical college hospital were considered for the study (200 MI patients and 150 age, sex matched controls). The genotype frequency in the population for L55M is LL=0.49, LM=0.40, MM=0.10, for Q192R is QQ=0.38, QR=0.46, RR=0.15 and it is consistent with Hardy-Weinberg equilibrium. The Logistic regression analysis showed MM vs LL having OR=1.44, M vs L OR=1.56 stating M allele is associated with developing risks of MI. In Q192R, RR vs QQ OR=3.25, R vs Q, OR=1.88 showing R allele having 3 times higher risk in developing MI.

Detection of resistance to fluoroquinolones and injectable drugs among antituberculosis drugs by allele-specific primer extension on a microsphere-based platform

Molecular drug susceptibility testing (DST) for antituberculosis drugs is important for improving the efficacy of multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) treatment. In this study, we developed a molecular high-throughput assay system based on allele-specific primer extension (ASPE) and MagPlex-TAG microspheres, referred to here as TAG-ASPE, which can detect mutations related to resistance to injectable second-line drugs and fluoroquinolones. Target genes were amplified by multiplex PCR using DNA from H37Rv and 190 clinical Mycobacterium tuberculosis strains and extended by ASPE using 22 ASPE primers. ASPE products were then sorted on the TAG-ASPE array and detected using a Luminex 200 system. The performance of the TAG-ASPE method was compared with that of sequencing and phenotypic DST. Comparison of the TAG-ASPE method with sequencing showed that the sensitivity and specificity of the TAG-ASPE method were 100% [95% confidence interval (CI), 96.38–100%] and 100% (95% CI, 95.70–100%) for the rrs gene and 100% (95% CI, 96.90–100%) and 100% (95% CI, 95.07–100%) for the gyrA gene, respectively. Compared with phenotypic DST, the sensitivity and specificity of the TAG-ASPE method for detecting drug-resistance mutations against injectable second-line drugs were 92.52% (95% CI, 85.8–96.72%) and 98.7% (95% CI, 92.98–99.97%), respectively. Additionally, the sensitivity and specificity for fluoroquinolone-resistance detection were 85.4% (95% CI, 78.36–90.85%) and 100% (95% CI, 92.38–100%), respectively. The results of this study demonstrate that the TAG-ASPE method can effectively detect mutations conferring resistance to second-line antituberculosis drugs in numerous clinical specimens.

Improved detection of BRAF V600E using allele-specific PCR coupled with external and internal controllers

Although traditional allele-specific PCR (tAS-PCR) is a common screening method for BRAF V600E mutations, its lower amplification specificity and mutation selectivity have limited its clinical applications. We hypothesize that these limitations are associated with the weaker specificities of allele-specific primers and the thermodynamic driving forces of DNA polymerase. We used three strategies to circumvent these limitations, namely, modifying allele-specific primers, introducing a competitive external allele-specific controller (i.e., cAS-PCR), and introducing a referenced internal positive controller in the cAS-PCR (i.e., rcAS-PCR). The amplification sensitivities and specificities were influenced by the position of the artificially introduced mismatched nucleotide in the allele-specific primers. Moreover, both cAS-PCR and rcAS-PCR could detect single-copy BRAF V600E alleles with higher mutation selectivity (0.1%) than tAS-PCR. In addition, cAS-PCR eliminated false-negative results caused by various PCR inhibitors that might be present in the DNA solutions. The rcAS-PCR could also be employed to avoid the false-negative results caused by low-abundance input templates in cAS-PCR. In conclusion, rcAS-PCR provides a rapid, simple, and low-cost method for detecting low levels of the mutated BRAF V600E gene.

The study of ABO genotyping for forensic application

ABO blood typing has been used for personal identification and paternity testing in forensic medicine and criminal investigations for many years, since the ABO blood types obtained from evidentiary samples are usually one of the initial pieces of information available that could lead to identification of the suspect. Because of this importance of the initial information, many different molecular typing methods have been reported to complement routine serological ABO blood typing in forensics. The method using a multiplex allele-specific primer set enables the detection of six SNP sites in the ABO gene (nt 261, 526 and 803) along with the determination of ABO genotypes from their combinations, which is applied to forensic science casework samples in this paper. An internal validation study has been conducted to show that this method successfully determined ABO genotypes of blood, blood stain, saliva stain, buccal swab, vaginal fluid, hair and low copy number sample with high sensitivity, accuracy, and reproducibility. These results suggest that the method using a multiplex allele-specific primer set is so reliable that it can be used as a rapid screen for forensic science casework samples before multi locus short tandem repeat (STR) profiling is performed.

Developing eight SNP-STR markers for DNA mixture detection

SNP-STR marker is a compound marker constituted by a STR marker and a SNP in the flanking sequence. SNP-STR marker has been introduced to improve the discrimination power of STR markers in previous reports. Recently, some researchers have reported that SNP-STR markers performed well in detecting unbalanced DNA mixtures by designing two allele-specific primers. In this study, STR markers were selected from previously reports which had a high polymorphism in Chinese Han population. Then SNP markers with minor allele frequency (MAF) above 0.10 and locate no more than 250bp from the repeat sequence of STR markers were screened from SNP database. Based on these criteria, eight SNP-STR markers were finally selected and amplification refractory mutation system (ARMS-PCR) method was used to achieve allele-specific amplification. 20 samples were used to estimate the polymorphism of SNPs in Chinese Han population. Six of the eight loci had a high polymorphism in SNP (above 0.20) which indicated the potential of discriminating DNA mixtures. Most of the primers target minor DNA at an excess of 100-fold major DNA. However, discrimination power is not high enough using these six loci. Therefore, further studies are necessary in developing more SNP-STR markers.

Identification of S genotypes in loquat (Eriobotrya japonica Lindl.) based on allele specific PCR

Loquat (Eriobotrya japonica Lindl) is an evergreen fruit tree and belongs to Maloideae in the Rosaceae, which carries the RNase-dependent gametophytic self-incompatibility system (GSI). Use of appropriate pollinator trees is essential for increasing fruit production. S-allele specific amplification system is an efficient and rapid method to identify S-genotypes. In this research, according to the sequences of S-RNase alleles, 9 specific primer pairs for amplifying S-RNase alleles (S2, S7, S6, S8, S9, S10, S11,S12 and S13) were designed, which located in the introns or in the areas with high polymorphism. The specificity of the 9 allele specific primer pairs was evaluated by eight loquat cultivars with known S-genotypes. After being verified their effectiveness, they were used to S-genotype 39 loquat cultivars from Longan and loquat nurseries in Fuzhou national fruit germplasm. The results showed that the 39 loquat cultivars were S-genotyped efficiently by allele specific PCR with the 9 primer pairs, in which 28 cultivars were identified for the first time and they were classified into 18 groups according to the genotype. The results also proved that the 9 S-allele specific primer pairs can efficiently identified S-genotype, which will be very useful for choosing pollinators or crossing parents in loquat production and breeding.

Single-Color Digital PCR Provides High-Performance Detection of Cancer Mutations from Circulating DNA

We describe a single-color digital PCR assay that detects and quantifies cancer mutations directly from circulating DNA collected from the plasma of cancer patients. This approach relies on a double-stranded DNA intercalator dye and paired allele-specific DNA primer sets to determine an absolute count of both the mutation and wild-type–bearing DNA molecules present in the sample. The cell-free DNA assay uses an input of 1 ng of nonamplified DNA, approximately 300 genome equivalents, and has a molecular limit of detection of three mutation DNA genome-equivalent molecules per assay reaction. When using more genome equivalents as input, we demonstrated a sensitivity of 0.10% for detecting the BRAF V600E and KRAS G12D mutations. We developed several mutation assays specific to the cancer driver mutations of patients' tumors and detected these same mutations directly from the nonamplified, circulating cell-free DNA. This rapid and high-performance digital PCR assay can be configured to detect specific cancer mutations unique to an individual cancer, making it a potentially valuable method for patient-specific longitudinal monitoring.

An investigation of a set of DIP-STR markers to detect unbalanced DNA mixtures among the southwest Chinese Han population

The resolution of DNA mixtures is still a difficult problem that is worthy of further study. A common method applied for analysing mixtures is the use of autosomal STR markers as well as related calculation software based on genotypes; however, these markers have a limitation in detecting minor DNA in unbalanced mixtures if major DNA constitutes over 95% of the stain. Novel biomarkers, such as Y-STR, DIP-STR and SNP-STR, have been shown to perform well in distinguishing DNA donors in this type of mixture. DIP-STR can successfully target minor DNA in 1000-fold background DNA using two separate allele-specific primers. However, whether this method can successfully detect minor DNA primarily depends on the distribution of the DIPs in a population. Until now, only Swiss population data have been reported; therefore in this study, we selected 10 DIP-STR markers that performed well in the Swiss population and investigated whether these markers were also useful among the southwest Chinese Han population. The allele frequencies were estimated based on 152 samples, and six of the ten DIP-STR makers had a relatively high probability of informative markers (I value), which indicated their potential usefulness in the southwest Chinese Han population. A comparative study of DIP-STR markers and autosomal STR markers demonstrated that DIP-STR markers detected minor DNA at a ratio of 1:1000, while autosomal STR markers often failed to genotype minor DNA because of strong background noises caused by large amount of major DNA. However, the discrimination power was not high enough using these six DIPs alone. Therefore, we suggest that development of a panel with more loci is imperative and that a panel combined with DIP-STR and SNP-STR markers may be a possible way to achieve better discrimination power.

Application of rapid PCR to authenticate Ranae Oviductus

Rapid allele-specific PCR primer was designed base on Cytb 155 A/T single nucleotide polymorphism, DNA was extracted by alkaline lysis and the PCR reaction systems including denatured and annealing temperature and cycle numbers were optimized. The results were performed to authenticate Ranae Oviductus and its 4 adulterants. When 100×SYBR Green I was added in the PCR product at 90 ℃ denatured 3 s, 62 ℃ annealing 20 s and 32 cycle. Ranae Oviductus visualized strong green fluorescence under 365 nm UV lamp whereas adulterants appeared negative. The whole process can be completed in 40 minutes．The established method provides the technical support for authentication of the Ranae Oviductus.