Alu and Poet Ankyrine Detection and Quantization in Cell Free Dna of Cancer Patients

Ahmed Darweesh Jabbar

doi:10.11648/j.crj.20180601.14

Abstract

In this study, specimens of plasma were collected from 96 from patient diagnosed with cancer from Al Karama teaching hospital. Also plasma collected from 25 healthy individual as control. Cell-free DNA (cfDNA) was extracted from 1ml plasma using high pure viral Nucleic Acid Kit as an alternative to high expensive cfDNA kit. cfDNA extracted according to the manufacturer’s instructions, but without the use of carrier RNA. This represent the first recorded method of using viral nucleic acid extraction kit for collection of cell free DNA. Results indicate that all cancer samples show significant increase of cfDNA in compare with control (p 0.01). and this dramatic increase in DNA concentration provide good indicator about health condition. DNA integrity have been checked depending on amplification of Arthrobacter luteus (Alu sequence). Both small (115) and large (247) large Alu stretches have amplified using RT PCR using SYBR green dye. Results shows that integrity of DNA extracted from patients and control are suitable for any further molecular investigation, and short Alu repeats are much less abundant in health individual in compare with cancer patient of all cancer types under study. Depending on these results one can conclude that almost all DNA obtained from normal patients is finely fragmented due to apoptosis action and that’s why give very little positive. results in compare with corresponding cancer patient which release large DNA fragments resulted from necrosis and NK/Tc cells activity, which is amplifiable more efficiently. In this work another molecular study conducted to investigate the cancer specific sequences. Since we deals with different cancers type in this study, two types of prostate, ovary, testis expressed protein (POTE Ankyrine) were tested 2α and 2β. Results shows that almost all healthy group were negative for specific POTE test while cancer patient samples are positive for POTE specific amplification. This results are promising since it could be developed to be a building block of cfDNA cancer tests.

Highlights

Cancer is a leading cause of death worldwide which needs appropriate diagnosis methods
Cell free DNA, or circulating cell free DNA, or cell free circulating DNA, isolated from body fluids such as plasma/serum/urine has emerged as an important tool for clinical diagnostics, The existence of cell free DNA in the human circulatory system has been known since the 1948, when Mandel and Matais managed to prove the existence of nucleic acids in human plasma [2]
DNA Extraction cf-DNA was extraction by viral nucleic acid kit III according to the manufacturer’s instructions and DNA stored in deep freezing till use, after that concentration of Cell-free DNA (cfDNA) measured by used nanodrob

Summary

Introduction

Cancer is a leading cause of death worldwide which needs appropriate diagnosis methods. Of circulating nucleotide acids including, circulating tumor DNA (ctDNA), circulating RNA or microRNAs, could be an ideal method for patients with cancer [1] Most of the nucleic acids (DNA and RNA) in the body are located within cells, but a fair amount of extracellular nucleic acids can be found circulating in the bloodstream called cell free DNA [2]. Plasma or serum are most frequently used for that purpose, the Cancer Research Journal 2018; 6(1): 20-25 presence of the free DNA was detected in urine [3], saliva [4], feces [5], synovial liquid [6], cerebrospinal fluid [7] and peritoneal fluid [8]. Majority of the free plasma DNA is doublestranded and consists of DNA molecules sized 0.18-21 kilo base [10]

Methods

Findings

Discussion

Conclusion