Fluorescent Primers Research Articles

Fluorescent primers amplification refractory mutation system qPCR (FP ARMS-qPCR) for MTHFR C677T SNP genotyping.

The currently used methods for the detection of methylene tetrahydrofolic acid reductase (MTHFR) C677T single nucleotide polymorphism (SNP) are either time-consuming or expensive. In this study, we devised an accurate, rapid and easy-to-use SNP detection system based on fluorescent primers amplification refractory mutation system qPCR, known as FP ARMS-qPCR. Fluorescent primers (FPs) modified by fluorescent dye or quencher near the 3' terminal thymine were designed. The reaction conditions were optimized and the performance was evaluated. Using commercial kits as controls, 242 samples were tested in parallel to verify the feasibility of the FP ARMS-qPCR assay. We demonstrated the good sensitivity and specificity of the FP ARMS-qPCR with optimized conditions. The assay was able to accurately distinguish between different SNP sites of MTHFR C677T in less than 2h using as low as 50 pg of template genomic DNA. Completely consistent genotyping results reveal that FP ARMS-qPCR is concordant with commercial kits. We established a specific, sensitive, and rapid FP ARMS-qPCR method for the detection of MTHFR C677T genotype. This could also serve as a potential diagnostic tool for a variety of diseases.

Analysis of Fluorescently Labeled ISSR Markers to Reveal the Genetic Diversity of Cassava Varieties (Manihot esculenta Crantz) in the Kerala’s Tribal Area of India

The study Inter Simple Sequence Repeat (ISSR) molecular markers to genotype distinct cassava (Manihot esculenta Crantz) cultivars within tribal communities in Kerala, India. Fluorescent ISSR primers were labelled with 6-carboxyfluorescein and used for PCR amplification. The total number of bands produced was 72, of which 49 were found to be polymorphic markers; an average of 17 polymorphic bands was obtained per primer. The manuscript is a scientific validation of the traditional knowledge of tribal people regarding the diversity and wealth of cassava crops in the region, and this validation can therefore help in protecting the farmers right on a particular landrace that they cultivate. The technique used in the diversity analysis is a time-saving process as compared to conventional breeding programs for crop improvement. The data in the manuscript also supports similarity and differences of each variety to one another, which is analysed statistically with the molecular marker-based data generated. By examining genetic diversity at a high resolution, ISSR markers can provide valuable insights into the evolutionary dynamics of these cassava populations. This research contributes to the broader goal of enhancing agricultural sustainability and food security in tribal communities.

Authenticity and adulteration identification of Bubali Cornu based on DNA fingerprints

Whether adulteration exists is a difficult problem in the identification of traditional Chinese medicine(TCM). Bubali Cornu is mainly available in the medicinal material market in the form of buffalo horn silk or buffalo horn powder but lacks obvious identification characteristics, so there is a risk of adulteration. However, the method of identification of adulteration in Bubali Cornu is lacking at present. In order to ensure authenticity and identify adulteration of TCM Bubali Cornu, control the quality of TCM Bubali Cornu, and ensure the authenticity of clinical use, the DNA fingerprints of 43 batches of samples from pharmaceutical companies and medicinal material markets were identified, and the amplification primers of fluorescent DNA fingerprints of Bubali Cornu and Bovis Grunniens Cornu were screened. The DNA fingerprints of Bubali Cornu were obtained by fluorescent capillary typing. The identification effect of fluorescent capillary typing on different adulteration ratios was also tested. Two pairs of fluorescent STR typing primers, namely 16Sa and CRc, which can distinguish Bubali Cornu and Bovis Grunniens Cornu, were screened out, and a DNA fingerprint identification method was established. The 16Sa migration peaks of Bovis Grunniens Cornu and Bubali Cornu were 223.4-223.9 bp and 225.5-226.1 bp. The CRc migration peaks of Bovis Grunniens Cornu and Bubali Cornu were 518.8-524.8 bp and 535.9-542.5 bp. The peak height of the migration peak could be used for preliminary quantification of the adulterants with an adulteration ratio below 50%, and the quantitative results were similar to the adulteration ratio. In this study, a simple and quick universal DNA fingerprint method was established for the identification of Bubali Cornu and its adulterants, which could realize the identification of TCM Bubali Cornu and the semi-quantitative identification of the adulterants.

A rapid and high-throughput multiplex genetic detection assay for detection, semi-quantification and virulence genotyping of Helicobacter pylori in non-invasive oral samples.

This study established a high-throughput multiplex genetic detection assay (HMGA) for rapid identification, semi-quantification and virulence analysis of Helicobacter pylori directly from the clinical non-invasive oral samples. The gastric mucosa and oral samples were collected from 242 patients in Shanghai from 2021 to 2022. All the samples were detected by routine clinical tests for H. pylori and Sanger sequenced for inconsistent results. A new multiplex PCR assay providing results within 4 hours was designed and optimized involving fluorescent dye-labeled specific primers targeted 16S rRNA gene, semi-quantitative gene ureC and 10 virulence genes of H. pylori. Semi-quantification was carried out by simulating the serial 10-fold dilutions of positive oral samples, and the H. pylori loads in different clinical samples were further compared. The mixed plasmids of virulence genes vacA s1, vacA m1 and vacA m2 were used to evaluate the performance on different genotypes. The consistency of 10 virulence genes in gastric mucosa, saliva, mouthwash and dental plaque of H. pylori-positive patients was compared. The non-invasive HMGA was highly specific for detection of all 12 targets of H. pylori and human internal reference gene β-globin, and the sensitivity to all target genes could reach 10 copies/μL. Compared with routine clinical tests and sequencing, non-invasive HMGA has a high level (>0.98) of sensitivity, specificity, accuracy, PPV, NPV and kappa coefficient for direct detection of H. pylori in oral samples. Moreover, by detecting peak area levels of ureC, it was confirmed that the H. pylori loads in gastric mucosa were significantly higher than those of the three kinds of oral samples (p<0.05). We also found that 45.0% (91/202) of patients had different H. pylori virulence genes in different oral samples. The concordance of positive detection rates of each virulence gene between saliva and gastric mucosa was more than 78% (p<0.05). The non-invasive HMGA proved to be a reliable method for the rapid H. pylori identification, semi-quantification and detection of 10 virulence genes directly in oral samples, providing a new idea for non-invasive detection of H. pylori.

High Frequency of Microsatellite Instability among Non-Metastatic Gastric Cancer.

Background: Microsatellite instability (MSI) is considered a key factor in carcinogenesis and a genetic alteration pattern in many types of cancers such as gastric cancer (GC). Although the role of MSI in colorectal cancer (CRC) is well known, its prognostic impact on GC has not been clearly defined. The assessment of MSI in GC has notbeen documented in the Iranian population yet. Therefore, this study analyzed the association of MSI status with GC in Iranian patients. Materials and Methods: We compared the frequency of MSI at 5 loci from formalin-fixed paraffin-embedded (FFPE) gastrectomy specimens, between metastatic and non-metastatic cases of GC (N = 60). A panel of five quasi-monomorphic markers and a single dinucleotide marker with linker-based fluorescent primers was used. Results: MSI was observed in 46.6% of cases, including MSI-high (H) (33.3%) and MSI-Low (L) (13.3%). Moreover, the most unstable and stable markers in our study were NR-21 and BAT-26 accordingly. MSI-H and MSI were seen more frequently in non-metastatic tumors (p= 0.028 and p= 0.019, respectively). Conclusion: The current study showed MSI status more frequently in non-metastatic GC which may reflect a good prognostic factor in GC like CRC. Although, larger and more comprehensive studies are needed to confirm this statement. A panel consisting of NR-21, BAT-25, and NR-27 mononucleotide markers appears to be reliable and useful markers for detecting MSI in GC in Iranian patients.

Abstract 1017: Impact of microsatellite instability status and immune scores on tumor after neoadjuvant chemoradiation therapy in locally advanced rectal cancer

Abstract Purpose: Neoadjuvant chemoradiation therapy (CRT) is a widely and effectively used preoperative treatment strategy in locally advanced rectal cancers. Tumors demonstrating microsatellite instability (MSI) are known to be infiltrated by higher numbers of lymphocytes and to be characterized by a more favorable prognosis as compared with tumors with intact repair systems, and MSI is the only potential biomarker to predict the outcome of immune checkpoint inhibitors. However, the relationship between MSI and response to neoadjuvant CRT and changes in MSI status after CRT in rectal cancer are not well understood. The aim of the present study was to investigate the MSI status before and after CRT and its association with CRT response in rectal cancer. Materials and methods: Formalin-fixed paraffin-embedded tissue specimens from pre-operative biopsy via sigmoidoscopy and surgical resection of the primary tumor were obtained from 84 rectal adenocarcinoma patients who underwent neoadjuvant CRT between March 2010 and May 2018. MSI assays were performed on DNA extracted from FFPE and matched normal tissues the extracted DNA was amplified by PCR with fluorescent dye-labeled primers targeting five microsatellite loci: BAT25, BAT26, D5S346, D2S123 and D17S250. Three immune biomarker scores were calculated from the 19 patients who were able to perform RNA sequencing analysis to compare the immune activities of the specimens between responder (n = 8) and non-responder (n = 11). The gene lists included in each score calculation were as follows: CXCL9, CXCL10, IDO1, IFNG, HLA-DRA, and STAT1; GZMA and PRF1; and CD247, CD2, CD3E, GZMH, NKG7, PRF1 and GZMK for the interferon-γ (IFN-γ) signature,1 the cytolytic activity2 and the immune signature,3 respectively. Results: Patients were classified, according to Mandard tumor regression grade (TRG), as responders (grade 1 or 2; 25 patients, 29.8%) and non-responders (grade 3, 4 or 5; 59 patients, 70.2%). The TRG of 84 patients was significantly different according to the pre-CRT MSI status (P = 0.024), but was not different according to the post-CRT MSI status among 60 patients who were eligible for the paired MSI test (P = 0.788). Changes in MSI status were observed in 11 of the 60 patients; MSS to MSI-low in 9 patients, MSS to MSI-high in 1 patient, and MSI-low to MSS in 1 patient. All three immune biomarker scores (the IFN-γ signature, the cytolytic activity and the immune signature) were higher in the responder group without significant difference (p = 0.536, p = 0.152, and p = 0.272, respectively). Conclusion: Our findings demonstrated that neoadjuvant CRT modulated the MSI status of rectal cancer and pre-CRT MSI status was significantly associated with response of CRT. Future studies on various immuno-profiles that can influence the CRT response using more samples are needed. Citation Format: Sung Uk Bae, Shin Kim, Sang Jun Byun, Hye Won Lee. Impact of microsatellite instability status and immune scores on tumor after neoadjuvant chemoradiation therapy in locally advanced rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1017.

Development of Multiplex PCR-based Protocols for Simultaneous Caterpillar Diagnosis of Three Spodoptera and One Mamestra Species (Lepidoptera: Noctuidae).

Since many noctuid moth species are highly destructive crop pests, it is essential to establish proper management strategies, which primarily require accurate and rapid species identification. However, diagnosis of noctuid species in the field, particularly at the larval stage, is very difficult due to their morphological similarity and individual color variation. In particular, caterpillars of Spodoptera exigua (Hübner), Spodoptera litura (Fabricius), Spodoptera frugiperda (Smith), and Mamestra brassicae (L.) (Lepidoptera: Noctuidae) are hard to be identified by morphology and frequently found on the same host crops in the same season, thus requiring a reliable species diagnosis method. To efficiently diagnose these species, we identified species-specific internal transcribed spacer 1 (ITS1) sequences and developed two molecular species diagnosis protocols using ITS1 markers. The first protocol was multiplex conventional PCR in conjunction with subsequent gel electrophoresis for species identification based on amplicon size. The second protocol was based on multiplex real-time PCR using fluorescent dye-labeled primers for single-step diagnosis. Template genomic DNA (gDNA) prepared by the DNA release method was also suitable for both protocols as the template prepared by DNA extraction. The two protocols enabled rapid and robust species diagnosis using a single multiplex PCR step. Depending on laboratory instrumentation, one of the two protocols can be easily adapted for species diagnosis of the four noctuid caterpillars in the field, which is essential for establishing proper management strategies. The multiplex real-time PCR protocol, in particular, will facilitate accurate diagnosis of the four species in a single step regardless of template gDNA quality.

Establishment of Multiplex Amplification System of STR Loci in Felis Catus and Its Forensic Application.

To construct a Felis catus STR loci multiplex amplification system and to evaluate its application value by testing the technical performance. The published Felis catus STR loci data were reviewed and analyzed to select the STR loci and sex identification loci that could be used for Felis catus individual identification and genetic identification. The fluorescent labeling primers were designed to construct the multiplex amplification system. The system was validated for sensitivity, accuracy, balance, stability, species specificity, tissue identity and mixture analysis, and investigated the genetic polymorphisms in 145 unrelated Felis catus samples. Sixteen Felis catus autosomal STR loci and one sex determining region of Y (SRY) were successfully selected, and constructed a multiplex amplification system containing the above loci. The complete profile of all alleles could still be obtained when the amount of DNA template was as low as 0.25 ng. There was no specific amplification peak in other common animal samples. Population genetic surveys showed that total discrimination power (TDP) of the 16 STR loci was 1-3.57×10-20, the cumulative probability of exclusion (CPE) was 1-6.35×10-5 and the cumulative probability of matching was 3.61×10-20. The Felis catus STR multiplex amplification system constructed in this study is highly sensitive, species-specific, and accurate in typing results, which can provide an effective solution for Felis catus species identification, individual identification and kinship identification in the field of forensic science.

New-Born Screening for Spinal Muscular Atrophy: Results of a Latvian Pilot Study

New disease-modifying treatments have recently been approved for 5q spinal muscular atrophy (SMA) and early treatment has been associated with a better clinical outcome. Accordingly, new-born screening (NBS) for SMA should be implemented to ensure early diagnosis of affected individuals. The aim of this study was to determine the feasibility and usefulness of NBS for SMA in Latvia. Between February and November of 2021, 10,411 parents consented to participation in the study. DNA testing for the SMN1 exon 7 homozygous deletion was conducted using qPCR with fluorescent locked nucleic acid primers. In the first month of testing, reporting of results took up to a maximum of 17 days after samples arrived in the laboratory. However, following familiarisation with the procedure, the median report time was reduced to 11 days after birth. Forty cases required samples to be taken again due to poor quality of the isolated DNA transpiring from either the quality of the blood punch or manual mistakes during DNA isolation. The SMN1 exon 7 homozygous deletion was identified in two individuals, which was subsequently confirmed by multiplex ligation-dependent probe amplification. When a NBS sample is taken 48 to 72 h after birth and transported to the laboratory within two working days after collection according to legal requirements, DNA test results can be reported to healthcare professionals before the 12th day of life. Expansion of our SMA 5q NBS procedure to the whole of Latvia is feasible and would facilitate early diagnosis and result in more effective treatment. We strongly advocate that SMA is added to the national Latvia Recommended Uniform Screening Panel.

MMR profile and microsatellite instability status in colorectal mucinous adenocarcinoma with synchronous metastasis: a new clue for the clinical practice

AimsMucinous adenocarcinoma (MA) is associated with a high frequency of microsatellite instability (MSI). In the metastatic setting, it is crucial to establish mismatch repair (MMR) and/or MSI status. However, genetic...

Genetic variability of dairy cattle breeds in Lithuania

Large numbers of different scientific studies are conducted to preserve the breeds and improve the existing ones by introducing the variability of the plate genes, which best shows the phenotypic characteristics that can improve the health of dairy cattle and the quality of their production. The main purpose of this study was to perform an analysis of genetic variability of Lithuanian cattle breeds. Three subpopulation groups were studied: Lithuanian Black and White (95 individuals), Lithuanian Red (49), and Lithuanian White and Red (48). Bovine genetic material was genotyped using a total of 11 fluorescent microsatellite primers to estimate genetic variability. All loci presented a high degree of polymorphism and a total of 292 different alleles (Na) were detected. Thirty-two private alleles were detected in all evaluated subpopulations. After completing pairwise population assignment, which is based on the distribution of allelic frequencies, three populations showed the tendency to group into three separate clusters. However, the performed Principal Coordinates Analysis (PCoA), which is based on genetic distances, showed no anticipated clear grouping. Bayesian structure analysis revealed three genetic clusters. Analysis of FST (0.001–0.027) and Nei genetic distance (0.029–0.084) revealed that the genetic diversity of inter subpopulation in cattle groups was estimated to be lower than the genetic diversity of intra subpopulation.

The Direct Semi-Quantitative Detection of 18 Pathogens and Simultaneous Screening for Nine Resistance Genes in Clinical Urine Samples by a High-Throughput Multiplex Genetic Detection System.

BackgroundUrinary tract infections (UTIs) are one the most common infections. The rapid and accurate identification of uropathogens, and the determination of antimicrobial susceptibility, are essential aspects of the management of UTIs. However, existing detection methods are associated with certain limitations. In this study, a new urinary tract infection high-throughput multiplex genetic detection system (UTI-HMGS) was developed for the semi-quantitative detection of 18 pathogens and the simultaneously screening of nine resistance genes directly from the clinical urine sample within 4 hours.MethodsWe designed and optimized a multiplex polymerase chain reaction (PCR) involving fluorescent dye-labeled specific primers to detect 18 pathogens and nine resistance genes. The specificity of the UTI-HMGS was tested using standard strains or plasmids for each gene target. The sensitivity of the UTI-HMGS assay was tested by the detection of serial tenfold dilutions of plasmids or simulated positive urine samples. We also collected clinical urine samples and used these to perform urine culture and antimicrobial susceptibility testing (AST). Finally, all urine samples were detected by UTI-HMGS and the results were compared with both urine culture and Sanger sequencing.ResultsUTI-HMGS showed high levels of sensitivity and specificity for the detection of uropathogens when compared with culture and sequencing. In addition, ten species of bacteria and three species of fungi were detected semi-quantitatively to allow accurate discrimination of significant bacteriuria and candiduria. The sensitivity of the UTI-HMGS for the all the target genes could reach 50 copies per reaction. In total, 531 urine samples were collected and analyzed by UTI-HMGS, which exhibited high levels of sensitivity and specificity for the detection of uropathogens and resistance genes when compared with Sanger sequencing. The results from UTI-HMGS showed that the detection rates of 15 pathogens were significantly higher (P<0.05) than that of the culture method. In addition, there were 41(7.72%, 41/531) urine samples were positive for difficult-to-culture pathogens, which were missed detected by routine culture method.ConclusionsUTI-HMGS proved to be an efficient method for the direct semi-quantitative detection of 18 uropathogens and the simultaneously screening of nine antibiotic resistance genes in urine samples. The UTI-HMGS could represent an alternative method for the clinical detection and monitoring of antibiotic resistance.

A Simple RFLP-Based Method for HFE Gene Multiplex Amplification and Determination of Hereditary Hemochromatosis-Causing Mutation C282Y and H63D Variant with Highly Sensitive Determination of Contamination.

Hereditary hemochromatosis is an autosomal recessive disorder with incomplete penetrance that results from excess iron absorption and can lead to chronic liver disease, fibrosis, cirrhosis, and hepatocellular carcinoma. The most common form of hereditary hemochromatosis in Western Europe is due to a homozygous mutation (p.(Cys282Tyr) or C282Y), in the HFE gene which encodes hereditary haemochromatosis protein. In the general European population, the frequency of the homozygous genotype is 0.4%, and this mutation explains up to 95% of hereditary hemochromatosis in France. We report here an improved PCR and restriction endonuclease assay based on multiplex amplification of HFE exon 4 (for C282Y detection), HFE exon 2 (for H63D detection), FZD1 gene (for digestion controls), and two Short Tandem Repeats (SE33 and FGA) for identity monitoring and contamination tracking. Fluorescent primers allow capillary electrophoresis, accurate allele tagging, and sensitive contamination detection.

Establishment of DNA molecular fingerprint of applied core germplasm in jute (&amp;lt;italic&amp;gt;Corchorus &amp;lt;/italic&amp;gt;spp.)

<p indent=0mm>It is important to construct and study the applied core collection in order to promote jute genetic breeding and explore excellent genes. In this study, the applied core collection in jute, including 61 accessions divided into 16 application-oriented features, such as high yield, high quality, disease resistance and so on, was established based on the field performance from 300 jute germplasm. 12 fluorescent core primers were screened from 46 pairs of core primers. Fluorescence labeled capillary electrophoresis was used to analyze the polymorphism of these 12 pairs of primers, and a total of 140 polymorphism sites were detected. The precise DNA molecular ID cards were constructed by the combination of the 12 core primer pairs from the data coded in the form of numbers and English letters. Bar code and quick response code DNA molecular were established and can be quickly scanned and recognized by electric gadget. These results could be beneficial to increase the application efficiency and rapid molecular identification in jute germplasm resources.

DNA Variations between Medicago truncatula Symbiotic Mutant Line and Native Variant Using Fluorescence-Based AFLP Marker

Genetic mutagenesis is a very efficient tool in studying genes function. Because of great benefits of legumes as human food and animal feed worldwide, we used a model plant Medicago truncatula for identification gene function related to nitrogen fixation process. Our mutant is a Medicago mutant line contains a tobacco Tnt1 retro-transposon mobile element with the two Long Terminal Repeats (LTR) inserted within the genome. Our mutant is predicted to contain a mutation in gene/s belonging to symbiotic interaction between legume and rhizobia. A novel technique was used based on using fluorescent oligonucleotide primers against oligonucleotide primers for Tnt1-LTRs of our mutant. This novel protocol was very successful in detection the polymorphism between our mutant line and the wild variant R108 using Biosystems 310 Genetic Analyzer. Electropherograms of the mutant line and wild type gave a total of 561 well- resolved AFLP peaks, 357of which were polymorphic peaks and 204 were monomorphic peaks. This novel technique enables the calculation percentage of polymorphism between the mutant line and the wild type. Additionally primers combinations amplified more bands from others to detect polymorphism between the plants

Loop-mediated isothermal amplification-fluorescent loop primer assay for the genotyping of a single nucleotide polymorphism at position 2254 in the viral DNA polymerase gene of equid alphaherpesvirus 1.

We developed a loop-mediated isothermal amplification (LAMP)-fluorescent loop primer (FLP) assay for genotyping the A/G2254 single nucleotide polymorphism (SNP) in the viral DNA polymerase gene of species Equid alphaherpesvirus 1 (EHV-1), which is associated with the neuropathogenic potential of this virus. In addition to the use of regular LAMP primers to amplify the target region, a 5'-FAM-labeled backward loop primer (FLB) and 3'-dabcyl-labeled quencher probe (QP) were designed for annealing curve analysis of the amplification product. The QP, which contacts the FLB, is located at the SNP site and has the A2254 allele. LAMP reactions were performed at 63°C for 40 min, and the subsequent annealing curve analyses were accomplished within 20 min. The LAMP-FLP assay could clearly differentiate A2254 and G2254 genotypes according to the difference in the annealing temperature of the QP between the 2 genotypes. Good agreement between the LAMP-FLP and the real-time PCR for genotyping of this SNP was observed in the detection of EHV-1 in equine clinical samples. The newly developed assay is a simple and rapid method for detecting and differentiating EHV-1 strains with A2254 and G2254 polymorphisms and would be suitable for clinical use.

Reduced False Positives and Improved Reporting of Loop-Mediated Isothermal Amplification using Quenched Fluorescent Primers

Loop-mediated isothermal amplification (LAMP) is increasingly used in molecular diagnostics as an alternative to PCR based methods. There are numerous reported techniques to detect the LAMP amplification including turbidity, bioluminescence and intercalating fluorescent dyes. In this report we show that quenched fluorescent labels on various LAMP primers can be used to quantify and detect target DNA molecules down to single copy numbers. By selecting different fluorophores, this method can be simply multiplexed. Moreover this highly specific LAMP detection technique can reduce the incidence of false positives originating from mispriming events. Attribution of these events to particular primers will help inform and improve LAMP primer design.

A multiplex SNP genotyping by allele-specificspecific PCR based on stem-loop and universal fluorescent primers of Chr1daxin mice.

Single nucleotide polymorphisms (SNPs) are one of the most common markers in mammals. Rapid, accurate, and multiplex typing of SNPs is critical for subsequent biological and genetic research. In this study, we have developed a novel method for multiplex genotyping SNPs in mice. The method involves allele-specific PCR amplification of genomic DNA with two stem-loop primers accompanied by two different universal fluorescent primers. Blue and green fluorescent signals were conveniently detected on a DNA sequencer. We verified four SNPs of 65 mice based on the novel method, and it is well suited for multiplex genotyping as it requires only one reaction per sample in a single tube with multiplex PCR. The use of universal fluorescent primers greatly reduces the cost of designing different fluorescent probes for each SNP. Therefore, this method can be applied to many biological and genetic studies, such as multiple candidate gene testing, genome-wide association study, pharmacogenetics, and medical diagnostics.

MSI-L/EMAST is a predictive biomarker for metastasis in colorectal cancer patients.

Microsatellite instability (MSI) is a prognostic marker in colorectal cancer (CRC). The biological significance of MSI-low (MSI-L) phenotype and its differences with microsatellite stable (MSS) phenotype remains unclear. The aim of this study is indicating the role of mononucleotide repeat in identifying MSI-L and revealing the association of MSI-L with elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) and oncologic outcome in CRC patients. MSI and EMAST status were analyzed using three quasimonomorphic panel (BAT-25, BAT-26, and NR-27) and five tetranucleotide repeats (D20S82, D20S85, D9S242, D8S321, and MYCL1), respectively, by capillary electrophoresis method without the need to fluorescent primers. The associations of MSI status with clinicopathological features, EMAST status, metastasis, and overall survival (OS) were investigated. Among 159 CRC patient 22.0% were MSI-H, 40.3% were MSS, 37.7% were MSI-L, and 41.5% showed EMAST + phenotype. MSI-L were associated with advanced stages, EMAST+ tumors and worse OS ( p ≤ 0.001). Metastasis was relatively common in MSI-L/EMAST + CRCs and BAT-25 were the most unstable marker in these tumors. MSI-L tumors have different clinicopathological features from MSS and MSI-H tumors. The MSI-L phenotype is a worse prognostic biomarker in CRC and when accompanied by EMAST could be a predictor for metastasis.

Development of nuclear microsatellite loci for Pinus albicaulis Engelm. (Pinaceae), a conifer of conservation concern.

Pinus albicaulis (whitebark pine) is a widely-distributed but rapidly declining high elevation western North American tree and a candidate for listing under the U.S. Endangered Species Act. Our objectives were to develop reliable nuclear microsatellite markers that can be used to assess within-population genetic diversity as well as seed and pollen migration dynamics, and to validate markers using two geographically proximal P. albicaulis populations. We identified 1,667 microsatellite-containing sequences from shotgun DNA libraries of P. albicaulis. Primer pairs were designed for 308 unique microsatellite-containing loci, and these were evaluated for PCR amplification success and segregation in a panel of diploid needle tissue. DNA was extracted with an SDS protocol, and primers were screened through gel electrophoresis. Microsatellites were genotyped through fluorescent primer fragment analysis. Ten novel and 13 transferred loci were found to be reproducible in analyses based on 20 foliage samples from each of two locations: Henderson Mountain, Custer Gallatin National Forest, Montana, and Mt. Washburn, Yellowstone National Park, Wyoming (USA). Transferred loci had higher numbers of alleles and expected heterozygosities than novel loci, but also revealed evidence for a higher frequency of null alleles. Eight of the 13 transferred loci deviated significantly from Hardy-Weinberg Equilibrium, and showed large positive FIS values that were likely inflated by null alleles. Mantel’s tests of transferred and novel markers showed no correlation between genetic and geographic distances within or among the two sampled populations. AMOVA suggests that 91% of genetic variability occurs within populations and 9% between the two populations. Studies assessing genetic diversity using these microsatellite loci can help guide future management and restoration activities for P. albicaulis.