Methylation analysis of cell-free DNA is a encouraging tool for tumor diagnosis, monitoring and prognosis. Sensitivity of methylation analysis is a very important matter due to the tiny amounts of cell-free DNA available in plasma. Most current methods of DNA methylation analysis are based on the difference of bisulfite-mediated deamination of cytosine between cytosine and 5-methylcytosine. However, the recovery of bisulfite-converted DNA based on current methods is very poor for the methylation analysis of cell-free DNA.
Results
We optimized a rapid method for the crucial steps of bisulfite conversion with high recovery of cell-free DNA. A rapid deamination step and alkaline desulfonation was combined with the purification of DNA on a silica column. The conversion efficiency and recovery of bisulfite-treated DNA was investigated by the droplet digital PCR. The optimization of the reaction results in complete cytosine conversion in 30 min at 70 °C and about 65% of recovery of bisulfite-treated cell-free DNA, which is higher than current methods.
Conclusions
The method allows high recovery from low levels of bisulfite-treated cell-free DNA, enhancing the analysis sensitivity of methylation detection from cell-free DNA.
Capillary column (320-m ID) liquid chromatography is an essential tool for the separation and concentration of low-picomole amounts of proteins and peptides for mass-spectrometric based structural analysis. We describe a detailed procedure for the fabrication of stable and efficient 50- to 180-m ID polyimide fused-silica columns. Columns were packed by conventional slurry packing with reversed-phase silica-based supports followed by column bed consolidation with acetonitrile and sonication. PVDF membrane or internal fused-silica particles were employed for column end-frit construction. The ability of these columns to withstand high back pressures (300–400 bar) enabled their use for rapid chromatography (>3400 cm/hr; i.e., 40 l/min for 200-m ID columns) and the loading of large sample volumes (up to 500 l). The accurate low flow rates (0.4–4.0 l/min) and precise gradient formation necessary to operate these columns were achieved by a simple modification of conventional HPLC systems [Moritz et al. (1992), J. Chromatogr.599, 119–130]. Column performance was evaluated for ability to resolve low-fmol amounts of all components of a mixture of PTH-amino acids and to separate peptides for on-line LC/MS analysis of peptide mixtures derived from in situ digestion of 2-DE resolved protein spots. 相似文献
Background: In order to ensure accurate survival estimates, population-based cancer registries must ascertain all, or nearly all, patients diagnosed with cancer in their catchment area, and obtain complete follow-up information on all deaths that occurred among registered cancer patients. In the US, linkage with state death records may not be sufficient to ascertain all deaths. Since 1979, all state vital statistics offices have reported their death certificate information to the National Death Index (NDI). Objective: This study was designed to measure the impact of linkage with the NDI on population-based relative and cancer cause-specific survival rates in the US. Methods: Central cancer registry records for patients diagnosed 1993–1995 from California, Colorado, and Idaho were linked with death certificate information (deaths 1993–2004) from their individual state vital statistics offices and with the NDI. Two databases were created: one contained incident records with deceased patients linked only to state death records and the second database contained incident records with deceased patients linked to both state death records and the NDI. Survival estimates and 95% confidence intervals from each database were compared by state and primary site category. Results: At 60 months follow-up, 42.1–48.1% of incident records linked with state death records and an additional 0.7–3.4% of records linked with the NDI. Survival point estimates from the analysis without NDI were not contained within the corresponding 95% CIs from the NDI augmented analysis for all sites combined and colorectal, pancreas, lung and bronchus, breast, prostate, non-Hodgkin lymphoma, and Kaposi sarcoma cases in all 3 states using relative survival methods. Additional combinations of state and primary site had significant survival estimate differences, which differed by method (relative versus cause-specific survival). Conclusion: To ensure accurate population-based cancer survival rates, linkage with the National Death Index to ascertain out of state and late registered deaths is a necessary process for US central cancer registries. 相似文献
Short tandem repeats (STRs) have been extensively used in forensic genetics. However, according to previous studies, the mutation rates of STRs are relatively high and are affected by many factors. Therefore, it is important to analyze STR mutations and determine the influence of underlying factors on STR mutation rates. Mutation rates of 28 autosomal STRs were determined from 8708 paternity testing cases in the Chinese Han population, and the relationships between STR mutation rates and population, sex, age, allele length and heterozygosity were investigated. A total of 279 mutations were observed at 27 loci in a total of 233,530 meiosis cases, including 273 (97.8%) one-step, 5 (1.8%) two-step and 1 (0.4%) three-step mutations. The overall average mutation rate was 1.19?×?10–3 (95% CI 1.06?×?10–3???1.34?×?10–3) ranging from 0 (TPOX) to 2.79?×?10–3 (D13S325). Mutation rate comparisons revealed statistically significant differences at several STRs among populations. Paternal mutations occurred more frequently than maternal mutations, at a ratio of 6.04:1, and the mutation rate tended to increase with paternal age. Moreover, our study revealed a bias towards contraction mutations for long alleles and expansion mutations for short alleles. No obvious bias was observed in the overall mutation direction. In addition, STR loci with higher expected heterozygosity (Hexp) tended to have higher mutation rates. This work revealed the relationships between STR mutation rates and several influencing factors, providing useful data and information for further research on STR mutations in forensic genetics.
Background: Cancer mortality statistics, an important indicator for monitoring cancer burden, are traditionally restricted to instances when cancer is determined to be the underlying cause of death (UCD) based on information recorded on standard certificates of death. This study's objective was to determine the impact of using multiple causes of death codes to compute site-specific cancer mortality statistics. Methods: The state cancer registries of California, Colorado and Idaho provided linked cancer registry and death certificate data for individuals who died between 2002 and 2004, had at least one cancer listed on their death certificate and were diagnosed with cancer between 1993 and 2004. These linked data were used to calculate the site-specific proportion of cancers not selected as the UCD (non-UCD) among all cancer-related deaths (any mention on the death certificate). In addition, the retrospective concordance between the death certificate and the population-based cancer registry, measured as confirmations rates, was calculated for deaths with cancer as the UCD, as a non-UCD, and for any mention. Results: Overall, non-UCD deaths comprised 9.5 percent of total deaths; 11 of the 79 cancer sites had proportions greater than 3 standard deviations from 9.5 percent. The confirmation rates for UCD and for any mention did not differ significantly for any of the cancer sites. Conclusion and impact: The site-specific variation in proportions and rates suggests that for a few cancer sites, death rates might be computed for both UCD and any mention of the cancer site on the death certificate. Nevertheless, this study provides evidence that, in general, restricting to UCD deaths will not under report cancer mortality statistics. 相似文献