Enhanced ascertainment of microchimerism with real‐time quantitative polymerase chain reaction amplification of insertion‐deletion polymorphisms

Lee, Tzong Hae; Chafets, Daniel M.; Reed, William; Wen, Li; Yang, Yunting; Chen, Jennifer; Utter, Garth H.; Owings, John T.; Busch, Michael P.

doi:10.1111/j.1537-2995.2006.00992.x

Cited by 47 publications

(73 citation statements)

References 20 publications

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“…24 Successful use of InDel amplification to detect microchimerism in blood transfusion recipients and hematopoietic stem cell replacement patients has been shown previously. 12,24,25 GAPDH primers were used as positive amplification controls [F 5 0 -GGACTGAGGCTCCCACCTTT -3 0 and R 5 0 -GCATGGACTGTGGTCTGCAA -3 0 ]. InDel sequences show a high degree of heterozygosity within the general population, therefore each individual will display a unique pattern of InDel sequence amplification.…”

Section: Real Time Pcrmentioning

confidence: 99%

“…It was determined that the sensitivity of reliable InDel amplification was 0.01% which is consistent with previously published assay reports. 24,25 To evaluate each participant sample, triplicate PCR reactions were conducted using 50 ng of total genomic DNA template in a 20mL PCR volume consisting of 1£ AmpliTaq Gold amplification buffer (Cat # N8080249; Applied Biosystems); 4.5 mM MgCl2 (Cat # AB-0359; Applied Biosystems); 200 nM each dNTP (Cat # BIO-39025; Bioline); 1.25mM Syto9 (Cat # S-3854; Life Technologies); 1 U AmpliTaq Gold DNA polymerase (Cat # N8080249; Applied Biosystems) and 500 nM of each primer (Geneworks). Real-time PCR was conducted using Rotor-Gene Q (Qiagen) with cycle conditions of 10 minutes at 95 C followed by 40 cycles of 30 seconds at 95 C, 15 seconds at 65 C, and 10 seconds at 72 C. Analysis was conducted using Rotor-Gene Q software version 2.2.3 (Qiagen).…”

Section: Real Time Pcrmentioning

confidence: 99%

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Leukodepleted blood components do not remove the potential for long-term transfusion-associated microchimerism in Australian major trauma patients

et al. 2014

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Section: Real Time Pcrmentioning

confidence: 99%

Section: Real Time Pcrmentioning

confidence: 99%

Leukodepleted blood components do not remove the potential for long-term transfusion-associated microchimerism in Australian major trauma patients

et al. 2014

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“…Total proviral HIV DNA was extracted from PBMCs by using modifications of previously described methods (34). This assay has an overall sensitivity of 1 copy/3 g of input DNA, equivalent to approximately 450,000 PBMCs (33,35). All proviral DNA levels were normalized to the input number of PBMCs.…”

Section: Methodsmentioning

confidence: 99%

Evidence for Persistent Low-Level Viremia in Individuals Who Control Human Immunodeficiency Virus in the Absence of Antiretroviral Therapy

Hatano

Delwart

Norris

et al. 2009

J Virol

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184

182

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A subset of antiretroviral-untreated, human immunodeficiency virus (HIV)-infected individuals are able to maintain undetectable plasma HIV RNA levels in the absence of antiretroviral therapy. These "elite" controllers are of high interest as they may provide novel insights regarding host mechanisms of virus control. The degree to which these individuals have residual plasma viremia has not been well defined. We performed a longitudinal study of 46 elite controllers, defined as HIV-seropositive, antiretroviral-untreated individuals with plasma HIV RNA levels of <50 to 75 copies/ml. The median duration of HIV diagnosis was 13 years, the median baseline CD4؉ T-cell count was 753 cells/mm 3 , and the median duration of follow-up was 16 months. Plasma and cellular HIV RNA levels were measured using the transcription-mediated amplification (TMA) assay (estimated limit of detection of <3.5 copies RNA/ml). A total of 1,117 TMA assays were performed (median of five time points/subject and four replicates/time point). All but one subject had detectable plasma HIV RNA on at least one time point, and 15 (33%) subjects had detectable RNA at all time points. The majority of controllers also had detectable cell-associated RNA and proviral DNA. A mixed-effect linear model showed no strong evidence of change in plasma RNA levels over time. In conclusion, the vast majority (98%) of elite controllers had measurable plasma HIV RNA, often at levels higher than that observed in antiretroviraltreated patients. This confirms the failure to eradicate the virus, even in these unique individuals who are able to reduce plasma viremia to very low levels without antiretroviral therapy.

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“…We describe here a novel application of CNVs, specifically CNDs, for measurement of the very low levels of fragmented DNA that are typically found in plasma. These deletion variants are present in all individuals and their use in this context offers significant advantages over existing allele-specific, hybridization-based techniques that target SNPs or short indels (3,4,12,13 ). Most importantly, since nonself DNA measurement using CNDs is made against a zero background, allele discrimination and, consequently, analytical sensitivity should be maximized.…”

Section: Discussionmentioning

confidence: 99%

Use of Copy Number Deletion Polymorphisms to Assess DNA Chimerism

et al. 2014

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BACKGROUND:We describe a novel approach that harnesses the ubiquity of copy number deletion polymorphisms in human genomes to definitively detect and quantify chimeric DNA in clinical samples. Unlike other molecular approaches to chimerism analysis, the copy number deletion (CND) method targets genomic loci (Ͼ50 base pairs in length) that are wholly absent from wild-type (i.e., self) background DNA sequences in a sex-independent manner. METHODS:Bespoke quantitative PCR (qPCR) CND assays were developed and validated using a series of DNA standards and chimeric plasma DNA samples collected from 2 allogeneic kidney transplant recipients and 12 pregnant women. Assay performance and informativeness were assessed using appropriate statistical methods. RESULTS:The CND qPCR assays showed high sensitivity, precision, and reliability for linear quantification of DNA chimerism down to 16 genomic equivalents (i.e., 106 pg). Fetal fraction (%) in 12 singleton male pregnancies was calculated using the CND qPCR approach, which showed closer agreement with single-nucleotide polymorphism-based massively parallel sequencing than the SRY (sex determining region Y) (Y chromosome) qPCR assay. The latter consistently underestimated the fetal fraction relative to the other methods. We also were able to measure biological changes in plasma nonself DNA concentrations in 2 renal transplant recipients. CONCLUSIONS:The CND qPCR technique is suitable for measurement of chimerism for monitoring of rejection in allogeneic organ transplantation and quantification of the cell-free fetal DNA fraction in maternal plasma samples used for noninvasive prenatal genetic testing. © 2014 American Association for Clinical ChemistryGenomic chimerism describes the coexistence of DNA or cells originating from more than one individual. The most frequently encountered examples are donorrecipient mixtures in individuals who have received an allogeneic organ transplant and fetal-maternal mixtures in the blood of pregnant women. Plasma DNA chimerism, which refers to the situation in which mixtures of "self" and "nonself" circulating cell-free DNA (ccfDNA) 9 are present in an individual's blood plasma, has recently acquired major clinical significance and application in noninvasive detection of fetal chromosome aneuploidy in pregnancy. In a research context, plasma DNA chimerism analysis promises new avenues for monitoring the immunological rejection of organ transplants.Quantitative assessment of the concentration of nonself plasma ccfDNA relies on distinct genetic differences between recipient and donor or mother and fetus. Copy number variants (CNVs) have not hitherto been used for this purpose. CNVs are losses or gains of segments of the genome, ranging from a few hundred base pairs to several megabases, which vary in copy number between individuals in a population (1 ). Genome analyses using massively parallel sequencing (MPS) and microarray technologies have revealed that 10%-15% of the genome is subject to copy number variation (2 ). The ubiquity, size, ...

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Enhanced ascertainment of microchimerism with real‐time quantitative polymerase chain reaction amplification of insertion‐deletion polymorphisms

Cited by 47 publications

References 20 publications

Leukodepleted blood components do not remove the potential for long-term transfusion-associated microchimerism in Australian major trauma patients

Leukodepleted blood components do not remove the potential for long-term transfusion-associated microchimerism in Australian major trauma patients

Evidence for Persistent Low-Level Viremia in Individuals Who Control Human Immunodeficiency Virus in the Absence of Antiretroviral Therapy

Use of Copy Number Deletion Polymorphisms to Assess DNA Chimerism

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