Methods for extracting and amplifying genomic DNA isolated from frozen serum

Dixon, Shannon C.; Horti, J.; Guo, Yi; Reed, Eddie; Figg, William D.

doi:10.1038/nbt0198-91

Cited by 38 publications

(22 citation statements)

References 10 publications

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“…However, it is important to notice that not all serum samples can be compared with each other without acknowledging these major issues during the preanalytic sample collecting and preparation. Different DNA extraction protocols may also contribute to the in vitro release of cell-free circulating DNA (29,30). Interestingly, a recent study by Umetani et al (31) showed that the higher amount of cell-free circulating DNA in serum compared with plasma is not caused by contaminated extraneous DNA released from leukocytes or other sources during extraction and seems negligible and concluded that serum is the better specimen source for circulating DNA as a biomarker.…”

Section: Discussionmentioning

confidence: 99%

Prognostic Value of Preoperative Serum Cell-Free Circulating DNA in Men with Prostate Cancer Undergoing Radical Prostatectomy

Bastian

Palapattu²,

Yegnasubramanian³

et al. 2007

Clinical Cancer Research

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Purpose: We evaluated the association of preoperative serum cell-free circulating DNA concentration in men with clinically localized prostate cancer who underwent radical prostatectomy with prostate-specific antigen (PSA) recurrence. Experimental Design: One hundred and ninety-two men with clinically localized prostate cancer, who underwent radical prostatectomy at the Johns Hopkins Hospital and had preoperative serum available for analyses constituted our study population. All serum samples were collected before prostate biopsy or at least 4 months after prostate biopsy. The total amount of serum cell-free circulating DNA from each sample was calculated using a standard curve generated via quantitative real-time PCR. PSA recurrence was defined as a single postoperative PSA level of z0.2. The natural logarithm (ln) of the DNA concentration was used for statistical analyses. Results: Of the 192 men in our study, 56 (29%) experienced PSA recurrence within the study period (median time to PSA recurrence 2 years). The median follow-up time for men free of disease at last follow-up was 3 years. The median serum cell-free DNA concentration of all men in the study was 5.3 ng/mL (mean 18.05 ng/mL; range 0.2-320 ng/mL). The mean serum DNA concentration for men who recurred and for those who did not was 3.8 F 34.1 and 13.7 F 33.6 ng/mL, respectively (P = 0.001). In a univariate analysis, ln DNA concentration was significantly associated with PSA recurrence (hazard ratio, 1.49; 95% confidence interval, 1.3-1.8; P < 0.001). In the multivariate model, ln DNA concentration was significantly associated with PSA recurrence (hazard ratio, 2.56; 95% confidence interval, 1.1-1.6; P = 0.003). Using bootstrap analyses, serum cell-free DNA concentrations z5.75 ng/mL were associated with an increased risk of PSA recurrence within 2 years of radical prostatectomy. Conclusion: Our study suggests that preoperative serum cell-free DNA concentration may be a useful prognostic biomarker for men with clinically localized prostate cancer treated with radical prostatectomy.

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Section: Discussionmentioning

confidence: 99%

Prognostic Value of Preoperative Serum Cell-Free Circulating DNA in Men with Prostate Cancer Undergoing Radical Prostatectomy

Bastian

Palapattu²,

Yegnasubramanian³

et al. 2007

Clinical Cancer Research

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“…However, the cost significantly varies from study to study. For Dixon et al (6), this kit is twice as expensive as other DNA isolation procedures. Löeffler et al (10), in a comparative study for extraction of DNA of C. albicans and Aspergillus niger cells from blood specimens, have found that QIAamp tissue kit is about 23 times more expensive than an in-house DNA isolation method.…”

Section: Vol 42 2004 Methods For Isolation Of Candidal Dna From Sermentioning

confidence: 99%

“…2.0 h for PCR amplification and analysis of data. All of these (6), in investigating a variety of methods for the extraction of genomic DNA from serum samples from prostate cancer patients, found that the QIAamp DNA blood kit reliably yielded pure, high-quality DNA for PCR. Wahyuningsih et al (16) used this kit to purify C. albicans DNA from serum specimens.…”

Section: Vol 42 2004 Methods For Isolation Of Candidal Dna From Sermentioning

confidence: 99%

Comparison of Different Methods of Isolation of DNA of Commonly Encountered Candida Species and Its Quantitation by Using a Real-Time PCR-Based Assay

et al. 2004

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Molecular diagnosis based on genomic amplification methods such as real-time PCR assay has been reported as an alternative to conventional culture for early detection of invasive candidiasis. However, a major limitation of the molecular method is the difficulty associated with breaking fungal cell walls since the DNA extraction step still requires more than half of a working day. It has been suggested that PCR detection of free template DNA in serum is preferred over the use of whole blood for the diagnosis of systemic candidiasis. In this study, two conventional procedures (the first [the HLGT method] consists of boiling sera in an alkaline guanidine-phenol-Tris reagent, and the second [the PKPC method] uses proteinase K digestion, followed by organic extraction) and three commercially available kits for DNA isolation were evaluated for sensitivity, purity, cost, and use of template for most clinically important Candida species in a TaqMan-based PCR assay. To optimize these procedures, we evaluated the effect of adding 0.5% bovine serum albumin to DNA extracts and found that it decreased the effects of inhibitors. The QIAamp DNA blood kit did significantly shorten the duration of the DNA isolation but was among the most expensive procedures. Furthermore, the QIAamp DNA blood kit proved to be as sensitive as the HLGT DNA isolation method for PCR amplification from 52 serum samples from hematology or oncology patients with clinically proven or suspected systemic Candida infections. All PCR-positive samples showed approximately the same Candida species load by both procedures (100% correspondence), whereas one discordant result was obtained between PCR and blood culture.The management of invasive fungal infections has been hampered by the inability to diagnose the infection at an early stage of disease. However, diagnosis remains difficult, since the only sign of infection may be a prolonged fever that is refractory to antibacterial treatment. In recent years, efforts have been made to develop molecular-biology-based methods for rapid diagnosis, which is crucial to the treatment and recovery of patients suffering from systemic candidiasis. In a comparison of the molecular diagnoses obtained by a real-time PCR-based method to the results of blood culture, the sensitivity and specificity of the molecular method with a C. albicans-specific probe observed with 122 clinical blood samples were 100 and 97%, respectively (12). However, a major limitation of the molecular method in comparison to blood culture was the difficulty associated with problems in breaking fungal cell walls since the DNA extraction step is still a limiting factor, requiring more than half of a working day.Actually, there is no consensus concerning the best blood fractions to be tested for diagnosis of systemic candidiasis. Several PCR methods have been developed for use either on whole-blood samples (7,11,12) or on serum samples (1, 3-5). However, in addition to being too time-consuming and laborintensive, protocols for extraction of cellular candi...

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“…Commercially available DNA isolation kits compared favourably to conventional DNA isolation methods in that situation [125]. In virtually all of the pertinent studies, an unknown [33][34] rather than a standardized DNA concentration was used for subsequent PCR amplification.…”

Section: Detection Of Circulating Tumor Nucleic Acids In Plasma/serummentioning

confidence: 99%

Noninvasive Molecular Detection of Cancer - the Bench and the Bedside

Goessl

2003

CMC

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The more profound understanding of the genetics and molecular pathways driving human tumorigenesis is paralleled by an ongoing interest to translate this knowledge into development of cancer biomarkers, termed molecular tumor markers. The molecular changes observed in tumors frequently constitute early events which are detectable as signatures of malignancy in body fluids and their occurrence may precede clinical cancer diagnosis. Thus, beyond applicability on tissue samples, molecular markers for tumor signatures should allow noninvasive or minimally invasive diagnosis in blood and/or other body fluid samples. However, to qualify as a clinically useful molecular tumor marker for initial diagnosis and detection of recurrent disease, a molecular tumor marker must have better test characteristics (sensitivity, specifity) than currently applied tumor markers. A molecular tumor marker should also be suitable for screening purposes by defining the subset of individuals for which definite cancer diagnosis by more invasive and/or expensive additional investigations is indicated. In addition, there is a demand for molecular tumor markers to be used as reliable surrogate endpoints in cancer prevention trials. Recommendation for the use of individual molecular tumor markers within evidence-based medicine criteria should ideally be derived from their overall efficacy to reduce tumor-specific mortality. This review focuses on DNA based, RNA based and proteomics based molecular methods of noninvasive cancer detection in bodily fluids and assess the value of these methods for the current and future clinical management of cancer patients.

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Methods for extracting and amplifying genomic DNA isolated from frozen serum

Cited by 38 publications

References 10 publications

Prognostic Value of Preoperative Serum Cell-Free Circulating DNA in Men with Prostate Cancer Undergoing Radical Prostatectomy

Prognostic Value of Preoperative Serum Cell-Free Circulating DNA in Men with Prostate Cancer Undergoing Radical Prostatectomy

Comparison of Different Methods of Isolation of DNA of Commonly Encountered Candida Species and Its Quantitation by Using a Real-Time PCR-Based Assay

Noninvasive Molecular Detection of Cancer - the Bench and the Bedside

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