Routine application of polymerase chain reaction in the diagnosis of monoclonality of B-cell lymphoid proliferations

We evaluated four polymerase chain reaction (PCR) methods for their efficiency in detecting monoclonality in a well-characterized panel of frozen and paraffin-embedded B-cell lymphoid proliferations. These approaches (referred to as FR3, FR3A, FR2, and FR1) are based on amplification of rearranged immunoglobulin heavy chain genes, using primers recognizing framework regions I, II, or III. FR3, FR3A and FR2 approaches reproducibly detected monoclonality in 51%, 72%, and 67% of DNAs from frozen lymphomas, respectively. No false-positives were observed. The combination of FR2 and FR3A methods raised the figure to 85%. Comparable results were obtained using paraffin-embedded lymphomas. Reproducibility of FR1 approach was unsatisfactory. The efficiency of all PCR approaches varied depending on lymphoma type. The highest detection rate was in small/intermediate cell and the lowest in centro-follicular lymphomas. Limiting dilution assays showed that PCR methods were able to detect monoclonal B-cell DNA representing 5% of nonlymphoid and 20% of polyclonal B-cell DNA. A diagnostic protocol may include quick and cost-effective PCR screening, particularly in cases of undetermined small cell lymphoid proliferations observed in fine needle aspirates or endoscopic biopsies. This would also reduce call-up of patients to obtain unfixed biopsies.     

Interactions between ovine cathepsin L, cystatin C and alpha 2-macroglobulin. Potential role in the genital tract

We prospectively investigated minimal residual disease (MRD) in 51 children with B-lineage acute lymphoblastic leukaemia (ALL) treated according to the Fralle 93 protocol. PCR follow-up was performed in children in morphological and cytogenetic complete remission, provided an immunoglobulin (IgH) gene rearrangement could be detected using FR 3/J(H) amplimers. MRD was studied according to our previously described methodology, with a few modifications including the use of a consensus J(H) probe to control for PCR efficiency variations. Out of the initial 51 patients, 34 were assessable for MRD at the end of induction at the time of analysis. MRD levels were as follows: > 1/10(3) in 26%, 1/10(3) to 1/10(4) in 50% and < 1/10(4) or not detectable in 24%. With a median follow-up of 20 months there were five relapses, all of which occurred in the group of patients with MRD > 1/10(3). To date, none of the patients with MRD < or = 1/10(3) (good molecular responder) has relapsed. Classification according to molecular response at the end of induction did not correlate with the conventional risks groups: there were no statistically significant differences between good and bad molecular responders. Of particular interest is the absence of correlation between WBC at diagnosis and MRD level at the end of induction. We conclude that classification of patients into good and bad molecular responders using PCR seems to be a better prognostic indicator than conventional risk factors in childhood B-lineage ALL. Patients with MRD level > 1/10(3) have a particularly poor outcome and should always be considered for alternative therapeutic strategies in the future, whereas in good molecular responders belonging to poor or intermediate risk categories, treatment de-escalation might be contemplated.     

Residual disease detection using fluorescent polymerase chain reaction at 20 weeks of therapy predicts clinical outcome in childhood acute lymphoblastic leukemia

PURPOSE: Ninety-five percent of children with acute lymphoblastic leukemia (ALL) will achieve a remission, but approximately 25% will relapse. Identifying these patients is difficult, as patients with adverse prognostic features at presentation are rare and the majority are standard risk. Analysis of minimal residual disease (MRD) may be able to determine those at risk of relapse, but the best method by which this can be accomplished has yet to be defined. The object of this study was to determine the predictive value of residual disease detection in a group of standard-risk patients with precursor-B ALL at a fixed point in therapy (week 20) using a simple fluorescent consensus immunoglobulin H (IgH) heavy chain polymerase chain reaction (PCR). PATIENTS AND METHODS: Forty-two patients who presented with precursor-B ALL with standard-risk clinical features and treated according to either the Medical Research Council (MRC) UKALL X or XI protocols were assessed using a combination of both fluorescent consensus framework I and framework III Ig heavy-chain PCR. The results of the PCR were analyzed on an ABI 373 gene sequencer with genescan software (Applied Biosystems, Foster City, CA). Clonal rearrangements detected at presentation were looked for at week 20. RESULTS: Of 42 patients, 35 had a clonal population detectable at presentation; of these, seven had more than two clonal rearrangements; this latter group showed a similar disease-free survival (DFS) to the group as a whole. Thirty of 35 patients were analyzed before their second course of intensification therapy at week 20. At this point, nine of 30 had a detectable clonal rearrangement, eight (89%) of whom have since relapsed with a median DFS of 27.5 months. Of the rest of the group (n=21), in whom no clonal rearrangement was detectable, only six (21%) have relapsed. CONCLUSION: Fluorescent IgH PCR at week 20 provides a sensitive and specific means to predict ultimate relapse (57% and 89%, respectively) and is a simple yet promising technique for the identification of patients at risk of poor outcome.     

Characterizing the target of acute stroke therapy

bcl-2/IgH fusion is considered a genetic error which occurs at the diversity (D) to joining (J(H)) stage of the gene rearrangement process in the immunoglobulin heavy chain (IgH) gene locus. Translocations of the bcl-2 protooncogene to the IgH locus at ontogenetically later IgH gene rearrangements are thought to represent exceptions. In the present study we analysed the junctional nucleotide sequence of 18 bcl-2/IgH fusion genes identifiable by polymerase chain reaction performed on DNA extracted from diagnostic lymph node tissue of 14 follicular lymphoma patients. In all clones studied, segments of variable length were found interposed between bcl-2 and J(H) gene sequences. Nucleotide sequence data analysis and comparisons performed with the corresponding germline sequences using the GenBank/EMBL database revealed the presence of D segments in most of the bcl-2/IgH fusion genes under study (13/18). By the same kind of computer-aided analysis, previously unrecognized D segments were identified in many published junctional sequences. These results suggest that bcl-2/IgH fusion events are very prevalent in rather more differentiated stages in B-cell ontogeny than previously recognized.     

Role of adenine nucleotides in the activation of microsomal cholesterol ester hydrolase by fructose or adenosine in rat hepatocytes

The significance of the demonstration of a clonal B-cell population in gastric lymphoid infiltrates was investigated by analysis of immunoglobulin heavy chain (IgH) gene rearrangements using sensitive polymerase chain reactions, employing fluorescently labelled primers to target the FR3 and FR1 regions. Tissue blocks were studied showing different histological features (high-grade lymphoma, low-grade lymphoma, and chronic gastritis) from 12 gastrectomies for primary gastric lymphoma, together with blocks showing chronic gastritis from 13 cases of gastric adenocarcinoma and biopsies from 33 patients with active Helicobacter-associated chronic gastritis. Clonal IgH gene rearrangements were detected in lymphoma samples from eight of the gastrectomies for lymphoma (67 per cent). In four of these eight specimens, clonal rearrangements were also detectable in the samples showing only chronic gastritis. Three of 28 (11 per cent) informative biopsies showing active Helicobacter-associated chronic gastritis had detectable clonal populations. Clonal rearrangements were also demonstrated in two of eight (25 per cent) informative blocks showing chronic gastritis from eight gastrectomies for adenocarcinoma. It is concluded that the detection of a clonal population in a suspicious lymphoid infiltrate does not confirm the diagnosis of lymphoma, nor does the absence of such a population imply benignity.     

Effects of bovine somatotropin (rbSt) concentration at different moisture levels on the physical stability of sucrose in freeze-dried rbSt/sucrose mixtures

Evaluation of minimal residual disease (MRD) in acute lymphoblastic leukaemia (ALL) is important for disease prognostication and early relapse detection. In this study, the rearranged third complementarity-determining-region (CDR-III) of immunoglobulin heavy chain (IgH) was used as a surrogate tumour marker for MRD evaluation. DNA obtained from marrows at diagnosis was amplified by the polymerase chain reaction (PCR) using a pair of consensus primers. After 2 rounds of DNA amplification and polyacrylamide gel separation, the nucleotide sequences of 87.5% (21/24) consecutive children with B-lineage ALL were obtained by automated sequencing. There were between 1-4 rearrangements per patient. Although the J5 and J6 joining regions were preferentially used, the rearranged sequences were unique for all 25 sequences obtained. Oligoprobes to the DNJ region were constructed and quantitation in 7 patients showed a detection sensitivity of 1 leukaemic cell in 10(4) to 10(5) normal cells compared to 3 in 100 using conventional morphological criteria. Serial bone marrow showed progressive decrease in the quantity of leukaemic cells, and no leukaemic sequences were detected during cessation of therapy in 4/7 patients. One patient with detectable MRD, absconded treatment and eventually relapsed. These results are consistent with the need to eliminate the leukaemic clones below MRD detection levels before the end of therapy at 2 years. In conclusion, this study describes a novel, simplified and sensitive method of MRD detection in childhood leukaemia.     

Splenic marginal zone cell lymphoma associated with clonal B-cell populations showing different immunoglobulin heavy chain sequences

Splenic marginal zone cell lymphomas (SMZCLs) are low-grade B-cell lymphomas that usually present with massive splenomegaly and subtle (subleukemic) peripheral blood involvement. Polymerase chain reaction (PCR) analysis of peripheral blood from a patient with subleukemic SMZCL showed evidence of two clonal immunoglobulin heavy chain (IgH) gene rearrangements. IgH PCR analysis of DNA derived from the patient's splenic neoplasm demonstrated a single clonal IgH rearrangement, which had a different electrophoretic mobility from either of the two PCR products detected in the patient's peripheral blood. Additional characterization of these PCR products by DNA sequencing demonstrated two independent IgH rearrangements in the peripheral blood, one of which used IgH joining region 6c (JH6C) and the other JH4. A different IgH rearrangement was present in the splenic tumor, which used JH4a. No sequences from the splenic neoplasm were detected in the peripheral blood and vice versa. This case illustrates that PCR might reveal monoclonal populations in peripheral blood unrelated to the presence of lymphoma in other anatomic compartments.     

Structure of Bcl-1 and IgH-CDR3 rearrangements as clonal markers in mantle cell lymphomas

Mantle cell lymphoma represent a clinicopathologically distinct entity of malignant non-Hodgkin's lymphoma (NHL) and are characterized by a specific chromosomal translocation t(11;14)(q13;q32) involving the cyclin D1 gene also designated as bcl-1/PRAD1 gene on chromosome 11 and the heavy chain immunoglobulin joining region on chromosome 14. We have established a PCR method to amplify t(11;14) junctional sequences in DNA from fresh frozen and paraffin-embedded tissue by bcl-1-specific primers in combination with a consensus immunoglobulin JH primer. A total of 65 cases histologically classified as mantle cell lymphoma (MCL) were analyzed for the presence of a t(11;14) translocation and monoclonal IgH-CDR3 rearrangements. From 26 patients with classical MCL and three cases with the anaplastic variant of MCL fresh frozen biopsy material was available for DNA extraction. We detected a bcl-1/JH rearrangement in 12 out of 29 samples (41%). In 36 cases paraffin-embedded lymph node tissue was the only source of DNA. In this material we found a bcl-1/JH rearrangement in six out of 31 samples with intact DNA (20%). To confirm the specificity of the PCR and to determine the bcl-1/JH junctional region sequences as clone-specific marker in individual patients we characterized the junctional DNA sequences by direct PCR sequencing in 16 cases. Interestingly we found that six bcl-1/JH junctions harbored DH segments in their N regions indicating that bcl-1/JH rearrangements can occur in a later stage of B cell ontogeny during which the complete VH to DH-JH joining or VH-replacement takes place. To investigate the suitability of IgH-CDR3 as sensitive molecular marker for those MCL patients in which a t(11;14) translocation can not easily be amplified, we additionally analysed 60 cases for the presence of monoclonally rearranged IgH genes by IgH-CDR3-PCR. A monoclonal IgH-CDR3 PCR product could be identified in 24 out of 29 fresh frozen samples (79%) whereas only 11 out of 31 samples (36%) with paraffin-derived DNA were positive. We demonstrate that automated fluorescence detection of monoclonal IgH-CDR3 PCR products allows the rapid and sensitive monitoring of minimal residual disease also in cases that lack a PCR amplifiable t(11;14) translocation. In combination with allele-specific primers the procedure may improve current experimental approaches for detection of occult MCL cells at initial staging and residual disease during and after therapy.     

The reliability of implant-retained hinging overdentures for the fully edentulous mandible. An up to 9-year longitudinal study

In B-cell malignancies, the uniqueness of the immunoglobulin heavy chain locus (IgH) clonal rearrangement provides a useful marker for the detection of minimal residual disease (MRD) after treatment. During the last decade, several techniques have been proposed and used for detecting MRD. In this review, we report the current PCR based techniques dealing with amplification of the VDJ segment since the CDR3 region is unique to each IgH rearrangement. The sensitivity of these techniques varies considerably with a detection level of one tumoral cell in 10(-2) to 10(-6) normal cells. Accurate and sensitive assessment of MRD may have profound impact in the clinical management of patients with hematologic malignancies. Although, a majority of studies have shown a good correlation between the rapidity or extent of the reduction in the number of tumoral cells and the subsequent relapse, other studies demonstrated substained positivity of PCR in patients in long term remission. Thus, current clinical studies of MRD should establish whether MRD predicts relapse uniformly and, therefore, justifies intensification of therapy in positive cases, or whether it simply detects leukemic cell populations whose proliferative potential has been altered by chemotherapy.     

Increased splanchnic blood flow after hypoglycaemia in diabetic and normal man: evidence against glucagon as a mediator

Analysis of non-Hodgkin lymphoma (NHL) involvement of bone marrow trephine biopsy specimens by morphologic features and immunohistochemistry is often difficult, and the criteria for involvement are ill defined. We compared the morphologic and immunohistochemical analysis of B-cell NHL involvement with immunoglobulin heavy chain gene (IgH) rearrangement analysis by polymerase chain reaction (PCR) amplification of the complementarity determining region 3 (CDR3) in bone marrow biopsy specimens from patients with mantle cell lymphoma (n = 53) or hairy cell leukemia (n = 71). By combing morphologic features and phenotype, 54 specimens were considered positive, 62 negative, and 8 inconclusive. PCR analysis showed clonal IgH rearrangements in 46 positive and 6 inconclusive specimens. No clonal IgH rearrangements were present in 61 negative specimens. The 1 false-positive and most false-negative PCR results were likely due to sampling error or DNA degradation of the fixed tissues. In most cases, bone marrow involvement by NHL can be identified by histologic and immunohistochemical examination. Furthermore, clonality of the B-cell population can be detected by amplification of the IgH CDR3 on DNA extracted from bone marrow trephine biopsy sections, which can be helpful in cases diagnosed as inconclusive.     

Trans-chromosomal recombination within the Ig heavy chain switch region in B lymphocytes

Somatic DNA rearrangements in B lymphocytes, including V(D)J gene rearrangements and isotype switching, generally occur in cis, i. e., intrachromosomally. We showed previously, however, that 3 to 7% of IgA heavy chains have the VH and Calpha regions encoded in trans. To determine whether the trans-association of VH and Calpha occurred by trans-chromosomal recombination, by trans-splicing, or by trans-chromosomal gene conversion, we generated and analyzed eight IgA-secreting rabbit hybridomas with trans-associated VH and Calpha heavy chains. By ELISA and by nucleotide sequence analysis we found that the VH and Calpha regions were encoded by genes that were in trans in the germline. We cloned the rearranged VDJ-Calpha gene from a fosmid library of one hybridoma and found that the expressed VH and Calpha genes were juxtaposed. Moreover, the juxtaposed VH and Calpha genes originated from different IgH alleles. From the same hybridoma, we also identified a fosmid clone with the other expected product of a trans-chromosomal recombination. The recombination breakpoint occurred within the Smicro/Salpha region, indicating that the trans-association of VH and Calpha genes occurred by trans-chromosomal recombination during isotype switching. We conclude that trans-chromosomal recombination occurs at an unexpectedly high frequency (7%) within the IgH locus of B lymphocytes in normal animals, which may explain the high incidence of B-cell tumors that arise from oncogene translocation into the IgH locus.     

Detection of residual disease in follicular lymphomas using the PCR technique: importance of clono-specific probes

Follicular lymphoma constitutes 30-40% of non-Hodgkin's lymphomas. Most patients have widespread disease at diagnosis. The clinical course is generally indolent, and it is not usually curable with available treatment. The source of relapse in patients who achieve complete clinical remission is residual neoplastic cells that are present below the limits of detection using standard techniques. With the development of PCR technology, the presence of these residual malignant cells [Minimal Residual Disease (MRD)] has been demonstrated clearly. Recently, an association of high-dose chemotherapy with autologous bone marrow or peripheral blood progenitor cell autograft appeared promising in the treatment of these lymphomas. In the search of clonal markers for the detection of MRD in follicular lymphomas, two strategies can be used. In the cases associated with the t(14;18) (q32;q21) chromosomal translocation, the bcl-2/JH junctional regions are amplified by PCR in approximately equal to 50% of cases and then sequenced in order to synthesize an anti-junction oligonucleotide probe specific for each patient's malignant clone (clonospecific probe). In the cases negative for this translocation, an alternative strategy consists in the amplification of immunoglobulin high chain (IgH) gene rearrangement (approximately equal to 75% of cases). The present review highlights the value of molecular markers such as bcl-2/JH and VH/JH rearrangements to follow the neoplastic clone and to detect MRD in patients with follicular lymphomas.     

Detection of p53 gene mutation in paraffin-embedded asbestos-related lung cancer tissue

The rearrangement of immunoglobulin heavy chain gene (IgH) and T cell receptor gamma gene (TcR gamma) was studied in 30 patients with acute lymphoblastic leukemia (ALL) by the polymerase chain reaction (PCR). 19 cases was found to have rearrangement of IgH gene, 12 of TcR gamma. Most of IgH rearrangement was characterized by one or two specific bands while some had more than two. Rearrangement of TcR gamma gene appeared as one specific band. A slight difference in number, size and lightness of bands was found among the patients. 4 different kinds of rearrangement were observed in the detection of IgH rearrangement in combination with TcR gamma gene. The rearranged patterns of IgH and TcR gamma gene as well as the clinical significance were discussed.     

Improved quantitation of minimal residual disease in multiple myeloma using real-time polymerase chain reaction and plasmid-DNA complementarity determining region III standards

The complementarity determining region III of the rearranged immunoglobulin heavy chain gene has been the target for tumor-specific PCR assays for the detection and follow-up of B-cell malignancies. Previously, these assays have relied on gel-based end point data collection methods (i.e., band densitometry) and, thus, have provided at best a semiquantitative assessment of tumor levels. We show the development of a novel, real-time TaqMan PCR assay to quantitate residual multiple myeloma cells in clinical samples after high-dose chemotherapy and autologous stem cell transplantation. We provide evidence that real-time PCR is reproducible, sensitive, and quantitative. In a 40-replicate PCR experiment targeting the beta-actin gene, the coefficient of variation for threshold cycle data was 1.6%, whereas it increased to 13.6% and 31%, respectively, for end point fluorescence and gel densitometry. Moreover, in an experiment directly comparing standard curves obtained from band densitometry and threshold cycle data, the standard curve constructed from threshold cycle data had a multiple R2 value of 1.00 and demonstrated a dynamic range >4 logs, compared with the 2-log linear range of gel densitometry. Finally, we show that when a complementarity determining region III-specific PCR primer is used in conjunction with a consensus primer for the immunoglobulin heavy chain joining gene, plasmid DNA can be used as a readily available and effective substitute for clonal plasma-cell genomic DNA when preparing standards. By applying real-time PCR to the analysis of clinical samples, we are able to quantitate levels of tumor involvement with unparalleled reproducibility and statistical confidence. Real-time PCR technology may well provide the accuracy and reliability necessary for minimal residual disease detection to have real prognostic significance.     

Design and synthesis of [111In]DTPA-folate for use as a tumor-targeted radiopharmaceutical

The diagnosis of early cutaneous T-cell lymphoma (CTCL) is a difficult point in dermatology. Recently, Southern blot analysis (SBA) and polymerase chain reaction (PCR) have been used to detect clonality in initial lesions in which clinical and histological findings are unspecific. Forty-one samples from 25 patients with CTCL were investigated for the presence of T-cell receptor-gamma gene rearrangement using a nested PCR technique and analysed by polyacrylamide gel electrophoresis (PAGE). Conventional SBA was also performed on 28 samples from 20 of these patients. In addition, 20 samples corresponding to patients with large plaque parapsoriasis (LPP), cutaneous B-cell lymphoma (CBCL) and eczema were analysed by PCR in the same way as were the CTCL specimens. Most of the CTCL specimens (81%) showed clonality on PCR analysis. Among patients with mycosis fungoides, 71% of initial patch lesions and 100% of plaques and tumours showed clonal disease. Clonality could be detected in three of four histologically negative post-treatment lesions. Clonal rearrangement was detected in one of three patients with LPP and in three of 10 patients with CBCL. None of the samples corresponding to patients with eczema showed positive results. SBA was significantly less sensitive than PCR in detecting clonality in CTCL patients (42% among early disease and 60% among advanced cases). The results indicate that this PCR/PAGE technique is a reliable and useful method for the detection of clonality in early skin lesions of CTCL patients and probably in the identification of silent extracutaneous involvement.     

TP53 DNA contact mutations are selectively associated with allelic loss and have a strong clinical impact in head and neck cancer

Recent studies have suggested that different mutation types within the core domain of the tumour suppressor protein p53, i.e. DNA contact mutations and structural mutations, confer different biological properties. We have analysed in 86 head and neck squamous cell carcinomas (HNSCC), whether these p53 mutation types have a differential clinical impact. Thirty-seven missense mutations were identified. Thirteen of these (36%) were DNA contact mutations, occurring in the L3 loop, in the H2 loop sheet helix motif, in the S10 beta strand and in Zinc binding residues. Microsatellite marker analysis revealed a selective association between these mutations and the loss of wild-type alleles (100% LOH vs 50% LOH in tumours with structural mutations; P=0.0034, Fisher's exact, 2-tailed). In comparison to structural mutations or to the absence of mutations in the core domain, DNA contact mutations were associated with higher tumour stages (84.6% vs 62%), a higher incidence of lymph node metastasis (91.7% vs 56%; P=0.014, Fisher's exact, 2-tailed), a shortened recurrence-free survival (8.1 months vs 23.7 months, P=0.047, log rank test) and overall survival (11 months vs 29.2 months; P=0.003, log rank test). The latter was also the case when only stage IV tumours were analysed (P=0.0055, log rank test). These data indicate that in HNSCC, TP53 DNA contact mutations confer a strong selection pressure to eliminate wild-type alleles, and that they result in an accelerated tumour progression and reduced therapeutic responsiveness.     

Rearrangement status of the malignant cell determines type of secondary IgH rearrangement (V-replacement or V to DJ joining) in childhood B precursor acute lymphoblastic leukemia

Immunoglobulin heavy chain (IgH) oligoclonality in childhood B precursor acute lymphoblastic leukemia (ALL) as determined by Southern analysis is found in 30-50% of patients and has been shown to be the result of ongoing IgH rearrangement (mostly V(H)-replacement and V(H) to D-J(H) joining) after malignant transformation. It is unknown however, what determines the type of secondary rearrangement. Also the biological basis of the variable degree of oligoclonality observed in childhood ALL is poorly understood. We analyzed in detail the IgH rearrangement status of the leukemic cells for a random panel of 18 childhood B precursor ALL patients by polymerase chain reaction (PCR)/sequencing analysis and by Southern analysis. By Southern analysis 10/18 (55.6%) patients were considered oligoclonal and 8/18 (44.4%) monoclonal. In contrast, by PCR minor clonal rearrangements were detected in 14/18 (77.8%) patients. V(H)-replacement was found in 7/14 patients, V(H) to D-J(H) joining in 6/14 patients and an unusual type of secondary rearrangement, V(H)-D to J(H) joining, in one patient. Only a single type of secondary rearrangement was detected in each patient. The type of secondary rearrangement (V(H)-replacement or V(H) to D-J(H) joining) depended on the rearrangement status (VDJ/VDJ or VDJ/DJ, respectively) of the dominant leukemic clone as determined by Southern analysis. We found that in addition to a more 'advanced' IgH rearrangement status patients with V(H)-replacements also have a more 'advanced' TCRdelta rearrangement status, which possibly reflects exposure of both the IgH locus and the TCRdelta locus to recombinase activity in a preleukemic clone. Finally, we investigated a putative relationship between oligoclonality by Southern analysis and S-phase fraction of the leukemic cell population. We found a significantly lower percentage cells in S-phase for oligoclonal patients as compared to monoclonal patients. Our data add to the understanding of ongoing rearrangement of antigen receptor loci in childhood ALL and have implications for the monitoring of minimal residual disease by PCR.     

Application of fluorescent dUTP on PCR and automated PCR product detection by using internal lane size standard

A convenient, simple, cost effective and efficient fluorescent PCR labeling for automated detection was developed by using fluorescent deoxyuridine triphosphate-(F)dUTP without changing any PCR reaction parameters. Multiple fluorescent (F)dUTPs in a broad range of concentration were tested on PCR reaction and then titration tests for optimum sample loading amount with automated detection were done by using Genescan 672 software on ABI 373A DNA sequencer. PCR product were automaticaly detected although the sensitivity of each color was different. Colors of (F)dUTP and (F)dUTP/dTTP ratio showed a significant influence on the incorpration of (F)dUTP in PCR reaction, so consequently the ratio gave a great impact on the PCR product detection results. To much incorpration of (F)dUTP changed the PCR fragment mobility and also sizing value. The application of (F)dUTP should have a great potential on linkage studies, mapping with microsatellite and mutation detection.