BACKGROUND: KRAS and BRAF genes are the biomarkers in Colorectal Cancer (CRC) which play prognostic and predictive roles in CRC treatment. Nowadays, the selection of rapid and available methods for studying KRAS and BRAF mutations in anti-EGFR therapy of patients suffering from CRC plays a significant role. In this study, the mutations of these two oncogenes were evaluated by different methods.
METHODS: This study was performed on 50 Formalin-Fixed Paraffin-Embedded (FFPE) tissue blocks of patients diagnosed with colorectal cancer. After DNA extraction, KRAS and BRAF gene mutations were evaluated using reverse dot blot, and results were compared with PCR-RFLP and allele-specific PCR for KRAS and BRAF mutations, respectively.
RESULTS: KRAS gene mutations were detected in 42% of patients, of which 30% were in codon 12 region, and 12% in codon 13. The most frequent mutations of KRAS were related to G12D and 10% of patients had BRAF mutated genes. The type of KRAS gene mutations could be evaluated by reverse dot blot method. In general, the results of PCR-RFLP and allele-specific PCR were similar to the findings by reverse dot blot method.
CONCLUSIONS: These findings suggest that PCR-RFLP and allele-specific PCR methods are suitable for screening the presence of the mutations in KRAS and BRAF oncogenes. In fact, another method with more sensitivity is needed for a more accurate assessment to determine the type of mutations. Due to higher speed of detection, reduced Turnaround Time (TAT), and possible role of some KRAS point mutations in overall survival, reverse dot blot analysis seems to be an optimal method.

Cancers are the most deadly diseases in the world and their incidences continue to increase over time. Particularly, breast cancer in females places 1(st) rank among other types of cancers in term of cancer cases (23%) and death incidence (14%). Recent findings support the correlation between (Ile)655(Val) SNP in the HER2 gene with breast cancer risk. Moreover, the (Ile)655(Val) HER2 gene polymorphism could be a predictive factor in a neoadjuvant therapy setting. Precise detection of the (Ile)655(Val) HER2 gene SNP in early breast cancer patients will be beneficial in designing the most suitable treatment and in increasing the efficacy of anticancer drugs. Here we develop a rapid and inexpensive method for (Ile)655(Val) SNP detection in the HER2 gene based on allele-specific PCR technology. Two forward primers and one common reverse primer were designed to anneal specifically either on the HER2 gene fragment containing the GG genotype or to the HER2 gene fragment containing the AA genotype where one of these primers had been added with poly-GC at 5\' upstream. Moreover, to increase discrimination level, mismatch bases at the SNP site and the 3(rd) base of each forward primers from 3\'end were added. To test the performance of the designed primers in discriminating a polymorphism and its annealing temperature, breast cancer specimen-derived genomic DNA (with GG genotype) and pGEM_HER2/AA (with AA genotype) were used as templates in the PCR reaction. The optimal annealing temperature for SNP detection was at 51.5°C as showed by the appearance of a 150 base pair (bp) band as AA genotype (pGEM_HER2/AA template), 116bp band as GG genotype (genomic DNA template), and both types of bands as AG genotype (mix of pGEM_HER2/AA and genomic DNA template). Allelic types of breast cancer patients were also determined using this optimized method compared to sanger sequencing. The 100% accordance was shown for all types of genotypes in both methods. The allele-specific PCR in this study may have application in determining polymorphisms of the breast cancers-originated (Ile)655(Val) HER2 gene.

Children with sickle cell disease (SCD) benefit from newborn screening, because life-threatening complications can be prevented by pre-symptomatic diagnosis. In Germany, the immigration of people from endemic countries is steadily growing. Comprehensive data about the epidemiology and prevalence of SCD in Germany are however lacking, and SCD is not included in the national newborn screening program. We provide data on the prevalence of SCD in a population from both urban and rural areas in Southwest Germany. Anonymized dried blood spots from 37,838 unselected newborns were analyzed by allele-specific PCR for the HbS mutation. Samples tested positive were subjected to Sanger sequencing of the entire β-globin coding sequence firstly to validate the screening and secondly to identify compound heterozygous SCD patients with other mutations of the β-globin gene. We identified 83 carriers of the sickle cell trait, three compound heterozygous SCD patients (two with sickle cell-β-thalassemia, one with sickle cell-Hb Tianshui) but no homozygous SCD patients. The novel molecular method and strategy for newborn screening for SCD presented here compares favorably in terms of sensitivity (1.0 for homozygous HbS, 0.996 for heterozygous HbS), specificity (0.996), practicability, and costs with conventional biochemical screening. Our results demonstrate a significant prevalence of SCD of approximately 1:12,000 in an unselected urban and rural population in Southwest Germany. Together with previously published even higher results from exclusively urban populations in Berlin and Hamburg, our data provide the basis for the decision on a newborn screening program for SCD in Germany.

Molecular epidemiology has transformed our knowledge of how tuberculosis (TB) is transmitted. Whole genome sequencing (WGS) has reached unprecedented levels of accuracy. However, it has increased technical requirements and costs, and analysis of data delays results. Our objective was to find a way to reconcile speed and ease of implementation with the high resolution of WGS. The targeted regional allele-specific oligonucleotide PCR (TRAP) assay presented here is based on allele-specific PCR targeting strain-specific single nucleotide polymorphisms, identified from WGS, and makes it possible to track actively transmitted Mycobacterium tuberculosis strains. A TRAP assay was optimized to track the most actively transmitted strains in a population in Almería, Southeast Spain, with high rates of TB. TRAP was transferred to the local laboratory where transmission was occurring. It performed well from cultured isolates and directly from sputa, enabling new secondary cases of infection from the actively transmitted strains to be detected. TRAP constitutes a fast, simple and low-cost tool that could modify surveillance of TB transmission. This pilot study could help to define a new model to survey TB transmission based on a decentralized multinodal network of local laboratories applying fast and low-cost TRAPs, which are developed by central reference centres, tailored to the specific demands of transmission at each local node.