Amplification of human minisatellites by the polymerase chain reaction: towards DNA fingerprinting of single cells

Jeffreys, Alec J.; Wilson, Victoria; Neumann, Rita; Keyte, John W.

doi:10.1093/nar/16.23.10953

Cited by 457 publications

(168 citation statements)

References 30 publications

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“…Based on the estimate of 6 pg of DNA per diploid cell, 23 we calculate that the lowest detection limit of our IgH PCR assay is 10 to 20 B cell genome equivalents. In other words, a limiting dilution down to one B cell will not be detectable by our assay, and hence will not yield a pseudomonoclonal band.…”

Section: Discussionmentioning

confidence: 99%

PCR Analysis of the Immunoglobulin Heavy Chain Gene in Polyclonal Processes Can Yield Pseudoclonal Bands as an Artifact of Low B Cell Number

Elenitoba-Johnson¹,

Bohling²,

Mitchell³

et al. 2000

The Journal of Molecular Diagnostics

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Polymerase chain reaction (PCR)-based analysis for detecting immunoglobulin heavy chain gene (IgH)rearrangements in lymphoproliferative disorders is well established. The presence of one or two discrete bands is interpreted as a monoclonal proliferation, whereas a smear pattern represents a polyclonal population. Prompted by our observation of discrete bands in histologically reactive processes with a relative paucity of B cells, we sought to determine whether low numbers of B cells in biopsy specimens could artifactually produce pseudomonoclonal bands. We performed IgH PCR analysis on serially diluted DNA samples from 5 B cell non-Hodgkin's lymphomas (B-NHLs), 5 reactive lymph nodes, 5 reactive tonsils and 10 microdissected germinal centers from a lymph node with follicular hyperplasia. We also assessed multiple aliquots of DNA samples from small biopsy specimens of reactive lymphocytic processes from the stomach (5 cases). PCR products were evaluated using high resolution agarose or polyacrylamide gels, and DNA sequencing was performed on IgH PCR products from two reactive germinal centers, which yielded monoclonal bands of identical size. The utility of molecular techniques such as Southern blot hybridization (SBH) analysis and polymerase chain reaction (PCR) in the assessment of clonality in lymphoproliferative disorders is well established. Although SBH is a fairly sensitive and specific method, its utility as a routine diagnostic tool is hampered by its requirement for high quality DNA, its labor-intensive nature, and its consequent long turnaround time. On the other hand, the rapid and less labor-intensive PCR continues to gain popularity as a technique for the assessment of clonality in lymphoproliferative disorders. 1,2 The results obtained with antigen receptor gene PCR correlate well with the results of SBH. [3][4][5] PCR-based assays have additional advantages over SBH analysis. PCR requires smaller quantities of DNA, and high molecular weight DNA is not necessary. Therefore, PCR has been applied to small biopsies and fixed paraffin-embedded tissues, which are generally unsuitable for SBH analysis. 6 PCR is extremely sensitive and can detect 1 positive cell in a background of 10 5 negative cells. Immunoglobulin heavy chain (IgH) PCR is capable of detecting 1 monoclonal B cell in a background of 10 2 to 10 3 polyclonal B cells. 7 The extreme sensitivity of PCR also constitutes a potential source for pitfalls in the interpretation of PCR-based antigen receptor gene rearrangement studies. Clearly, contamination is a frequent concern. Additionally, we have observed discrete bands in samples obtained from small biopsy specimens in which histological and immunophenotypic evaluation revealed a reactive process. We believe that the discrete bands generated in this situation are related to the paucity of B

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Section: Discussionmentioning

confidence: 99%

PCR Analysis of the Immunoglobulin Heavy Chain Gene in Polyclonal Processes Can Yield Pseudoclonal Bands as an Artifact of Low B Cell Number

Elenitoba-Johnson¹,

Bohling²,

Mitchell³

et al. 2000

The Journal of Molecular Diagnostics

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“…Each result was then converted into estimates of the actual amounts of amplifiable gDNA in mg and multiplied by 3.3 Â 10 5 to estimate the number of amplifiable haploid genomes. 22,23 PCRs were performed using PrimerDesign (Southampton, UK) master mix under standard conditions, with 5 ml of gDNA (approximately 100 ng). PCRs were set up on a RotorGene CAS1200 robot and run on the RotorGene 6000 platform (Cambridge, UK).…”

Section: Absolute Quantification Of Gdna Fusions In Pre-imatinib Samplesmentioning

confidence: 99%

Detection and molecular monitoring of FIP1L1-PDGFRA-positive disease by analysis of patient-specific genomic DNA fusion junctions

et al. 2008

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To evaluate current detection methods for FIP1L1-PDGFRA in hypereosinophilic syndrome (HES), we developed a means to rapidly amplify genomic break points. We screened 202 cases and detected genomic junctions in all samples previously identified as RT-PCR positive (n ¼ 43). Genomic fusions were amplified by single step PCR in all cases whereas only 22 (51%) were single step RT-PCR positive. Importantly, FIP1L1-PDGFRA was detected in two cases that initially tested negative by RT-PCR or fluorescence in situ hybridization. Absolute quantitation of the fusion by real-time PCR from genomic DNA (gDNA) using patient-specific primer/probe combinations at presentation (n ¼ 13) revealed a 40-fold variation between patients (range, 0.027-1.1 FIP1L1-PDGFRA copies/haploid genome). In follow up samples, quantitative analysis of gDNA gave 1-2 log greater sensitivity than RQ-PCR of cDNA. Minimal residual disease assessment using gDNA showed that 11 of 13 patients achieved complete molecular response to imatinib within a median of 9 months (range, 3-17) of starting treatment, with a sensitivity of detection of up to 1 in 10 5 . One case relapsed with an acquired D842V mutation. We conclude that detection of FIP1L1-PDGFRA from gDNA is a useful adjunct to standard diagnostic procedures and enables more sensitive follow up of positive cases after treatment.

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“…PCR amplification of five different minisatellite loci were used to analyze of MC (D1S80, 28 D11S533, 29 IGHJ, 30 33.6 31 and TCRAD 32 ) in the initial screening process before SCT, to find an informative locus separating the donor and the recipient. Data concerning the VNTR primers used in this study are shown in Table 2.…”

Section: Vntr Analysismentioning

confidence: 99%

Leukemia lineage-specific chimerism analysis is a sensitive predictor of relapse in patients with acute myeloid leukemia and myelodysplastic syndrome after allogeneic stem cell transplantation

et al. 2001

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One of the major complications after allogeneic stem cell transplantation (SCT) in patients with malignant disease is a high frequency of relapse. We have prospectively analyzed the clinical impact of recipient-derived chimeric cells in 30 patients with acute myeloid leukemia and myelodysplastic syndrome after SCT. In order to improve sensitivity and specificity, all samples were cell-separated by using immunomagnetic beads according to the patient's leukemia phenotype, expressed at diagnosis or relapse before SCT. Twelve out of 30 patients experienced a clinical relapse after SCT. Median follow-up time for patients alive and without relapse (n = 15) was 30 (16-47) months. Mixed chimerism in peripheral blood (PB) and bone marrow (BM) у1 month post SCT, in the leukemia-affected cell lineage, was detected in 14/30 patients. Ten of these 14 patients relapsed as compared to 2/16 with donor chimerism (DC) (P Ͻ0.01). All eight patients with MC in peripheral blood у1 month after SCT relapsed vs 4/22 DC patients (P Ͻ 0.001). MC was detected a median of 66 (23-332) days before hematological relapse. No correlation was found between T cell MC and relapse. In this study, chimerism analysis showed a higher sensitivity and specificity vs morphological examination. In conclusion, this study may provide a rational basis for treatment with adoptive immunotherapy at an earlier time after SCT than at present, in patients with AML and MDS, in order to treat recurrences of malignant disease. Leukemia (2001Leukemia ( ) 15, 1976Leukemia ( -1985

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Amplification of human minisatellites by the polymerase chain reaction: towards DNA fingerprinting of single cells

Cited by 457 publications

References 30 publications

PCR Analysis of the Immunoglobulin Heavy Chain Gene in Polyclonal Processes Can Yield Pseudoclonal Bands as an Artifact of Low B Cell Number

PCR Analysis of the Immunoglobulin Heavy Chain Gene in Polyclonal Processes Can Yield Pseudoclonal Bands as an Artifact of Low B Cell Number

Detection and molecular monitoring of FIP1L1-PDGFRA-positive disease by analysis of patient-specific genomic DNA fusion junctions

Leukemia lineage-specific chimerism analysis is a sensitive predictor of relapse in patients with acute myeloid leukemia and myelodysplastic syndrome after allogeneic stem cell transplantation

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