Measurement Of DNA Degradation Kinetics In Serum, Urine And Saliva Based On Automatic STR Analysis System

Background:Circulating DNA is the DNA free from cells existing in the boidly fluids, such as:serum, plasma, saliva, urine, cerebrospinal fluid (CSF), prostatic secretious. Becauseof its significant values in clinical diagnosis for tumor, myocardial infarction andprenatal diagnosis, circulating DNA detection also has the name of “liquid biopsy”.However, circulating DNA is naturally degraded in bodily fluids, the detection ofwhich is a multiple process including sample collection, storage all the way toanalysis; each step can greatly impact the final results. By analyzing20short tandemrepeats (STR), located on16different autosomes and sex chromosome, this paper isto analyze DNA degradation to fragments with lengths ranging from77-446bp inserum, urine and saliva under different temperatures and the degradation effects ofurine to DNA, so that the impact of the storage time and condition between bodilyfluid samples collection and extraction, and the amplicon size to the detection resultsare discussed.Methods:“Naked DNA” and DNA-protein complexes were prepared, added to the samplesto be analyzed and incubated at room temperature (RT) and37°C for various lengthsof time. Automated DNA purification system based on magnetic nanoparticles wasused for DNA extraction from incubated samples. And alleles of20STR loci wereamplifed by using paternity test technology in a forensic laboratory. Following this approach, we analyzed the temporal-degradation patterns of naked DNA andDNA-protein complexes in serum, urine and saliva. Meanwhile, to research on theeffects of enzymes and thermostable components of urine on DNA degradation, urinewas separately processed by adding EDTA and high temperature high pressure, andurea solution of the similar concentration to that of urine was prepared.Results:1. Kinetics of DNA degradation in serumThe degradation of naked DNA and DNA-protein complexes followed afirst-order clearance model in serum under RT and37°C. The half-lives were asfollows: in RT: the half-lives of naked DNA and DNA-protein complexes in serumwere71.4min and346.5min, respectively; in37°C, the half-lives were26.8min and157.5min, respectively.2. Kinetics of DNA degradation in salivaThe degradation of saliva DNA followed a first first-order clearance model underRT and37°C, with the value of253.2min and172.9min, respectively.3. Effects of urine on DNA stabilityThe degradation impact of urine to DNA was extremely strong, so the diluentsmultiples for half-quantity of added DNA degradation were determined to quantifythe degradation effects of urine on DNA. Half of the added “naked DNA” andDNA-protein complexes became degraded at dilution factors of6.9and4.4,respectively.4. Preliminary analysis of urine components leading to DNA degradationThermostable components of urine played a vital role in DNA degradation, onwhich the urea also had some effects, while EDTA can partially inhibit thedegradation effect of urine.Conclusions:1.“Naked DNA” and DNA-protein complexes followed a first-model clearancein serum, with the half-lives in RT were about1h and6h, respectively; and0.5h,1hin37°C, respectively.2. The kinetics of saliva DNA followed a first-order clearance model. The half-lives under RT and37°C were approximately4h and3h, respectively.3. Compared with serum and saliva, urine had a strongest effect on DNAdegradation. And the degradation effect may be related to the thermostable andthermolabile components of urine.