cell free DNA.ppt

1、Circulating Cell Free DNA (CCFDNA),a liquid biopsy,DNA as a biomarker for early diagnosis, prognosis and monitoring of therapy. The DNA : normal locations (nucleus and mitochondria)or circulating free. Circulating cell free DNA (CCFDNA) molecules were first identified in 1948. Extracellular nucleic

2、acids:cell-free (complexes with proteins ) or bound to the cell-surface(membrane-bound particles) The mean quantity of plasma circulating DNA in normal subject is varied from less than 10 ng/mL to more than 1500 ng/mL.,a liquid biopsy,non-invasive diagnostic evaluation high accuracy reasonable cost

3、when repeated tumor biopsies are not feasible and genomic analysis of archival tumor is deemed insufficien.,The characters of ccfDNA,quantity :ccfDNA to be present in higher levels among patients with autoimmune diseases and cancer as compared with healthy individuals quality : To date, the majority

4、 of the gene sequences of ccfDNA associated with disease (e.g., p53, the Ras family, beta-globin, or beta-actin) are not part of circulating DNA in healthy individuals. Most of the plasma DNA of normal individuals belongs to the Alu repeat family.,The Source of DNA in Circulation,Apoptosis(180-1000b

5、p) Necrosis (mainly 10,000 bp) spontaneous release of newly synthesized nucleic acids break down of any pathogens In healthy individuals, the concentration of circulating DNA is low. In cancer patients,the accumulation of ccfDNA : excessive release of DNA caused by massive cell death and inefficient

6、 removal of the dead cells.,Two main mechanisms,for the release of free-circulating nucleic acids : passive:release of DNA from proliferating cells , exosome production , the leakage after tumor necrosis or apoptosis and the lysis of circulating tumor cells(CTC) or micrometastases.Macrophages and ph

7、agocytes play an important role in phagocytosis of necrotic and apoptotic cells and can release digested nucleic acids into the microenvironment. active:one potential explanation hypothesizes that cancer cells release nucleic acids to transform the targeted recipient cells at distant locations,Obtai

8、ning DNA from Circulation,Detection for CCFDNA,Tumor-derived CCFDNA exhibits a specific profile based on DNA size and significantly higher DNA fragmentation . he detection of point mutations, chromosomal alterations (inversion/ deletion.) Cancer Related DNA Methylation; Microsatellite Alterations:Lo

9、ss of Heterozygosity (LOH) and Microsatellite Instability (MSI),the advantages of ccfDNA,non-invasive diagnostic evaluation high accuracy reasonable cost when repeated tumor biopsies are not feasible and genomic analysis of archival tumor is deemed insufficien. assessing therapy response,conventiona

10、l protein markers(such as CA-125, CA199 and CEA), have common limitations owing to their low sensitivity and specificity follow up disease progression,use of DNA assays for clinical medicine can be significantly sensitive and specific if cancer-specific DNA alterations are tested instead of elevatio

11、n of circulating DNA concentration,Circulating cell-free DNA (CCFDNA) has been suggested as a cancer biomarker. Several methods have been implemented to determine the quantitative and qualitative tumor-specific alterations of (CCFDNA), such as DNA strand integrity, gene amplification, gene mutations, gene methylation and microsatellite abnormalities as diagnostic, prognostic, and monitoring ma