DNA and its types
Journal of Pharmacology and Therapeutic Research is an open access, peer reviewed journal that focuses on the interdisciplinary research offering therapeutic solutions to various neurological, genetics, psychological, and respiratory issues affecting the human beings.
Circulating free DNA (cfDNA) are degraded DNA fragments released to the blood plasma. cfDNA can be used to describe various forms of DNA freely circulating the bloodstream, including circulating tumor DNA (ctDNA) and cell-free fetal DNA (cfDNA). Elevated levels of cfDNA are observed in cancer, especially in advanced disease. CfDNA has been shown to be a useful biomarker for a multitude of ailments other than cancer and fetal medicine. This includes but is not limited to trauma, sepsis, aseptic inflammation, myocardial infarction, stroke, transplantation, diabetes, and sickle cell disease. cfDNA is mostly a double-stranded extracellular molecule of DNA, consisting of small fragments (70 to 200 bp) and larger fragments (21 kb) and has been recognized as an accurate marker for the diagnosis of prostate cancer and breast cancer.
Collection and purification cfDNA purification is prone to contamination due to ruptured blood cells during the purification process. Because of this, different purification methods can lead to significantly different cfDNA extraction yields. At the moment, typical purification methods involve collection of blood via venipuncture, centrifugation to pellet the cells, and extraction of cfDNA from the plasma. The specific method for extraction of cfDNA from the plasma depends on the protocol desired.
Analysis of cfDNA in Plasma In general, the detection of specific DNA sequences in cfDNA can be done by two means; sequence specific detection (PCR based) and general genomic analysis of all cfDNA present in the blood (DNA sequencing). The presence of cfDNA containing DNA from tumor cells was originally characterized using PCR amplification of mutated genes from extracted cfDNA. PCR based analysis of cfDNA typically rely on the analytical nature of qPCR and digital PCR. Both of these techniques can detect down to a single targeted molecule present in a sample. For this reason the PCR based method of detection is still very prominent tool in cfDNA detection. This method has the limitation of not being able to detect larger structural variant present in ctDNA and for this reason massively parallel next generation sequencing is also used to determine ctDNA content in cfDNA.
Massively Parallel Sequencing Massively parallel sequencing (MPS) has allowed the deep sequencing of cfDNA. This deep sequencing is required to detect mutant ctDNA present in low concentrations in the plasma. Two main sequencing techniques are typically used for analysis of mutant cfDNA; PCR amplicon sequencing and hybrid capture sequencing. Other forms of genetic alterations can be analysed using ctDNA (e.g. somatic copy number alterations or genetic rearrangements). Here, methods based on untargeted sequencing, like WGS or low coverage WGS, are mainly used.
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