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R Luthra

Publications and source records attributed to R Luthra.

At least 37 records · Page 2Linked to original sources

Novel 5' exonuclease-based real-time PCR assay for the detection of t(14;18)(q32;q21) in patients with follicular lymphoma.

The exonuclease-based real-time polymerase chain reaction (PCR) exploits 5'-->3' exonuclease activity of Taq polymerase and measures PCR product accumulation as the reaction proceeds through a dual-labeled fluorogenic probe. The utility of this exonuclease-based PCR assay as a rapid alternative to conventional PCR for follicular lymphoma-associated t(14;18)(q32;q21) was evaluated in this study. The specificity of the assay for t(14;18) involving bcl-2 and immunoglobulin heavy-chain joining region (JH) genes was assessed by analyzing DNA from 53 patients (38 B-cell non-Hodgkin's lymphomas and 15 nonneoplastic proliferations) and correlating the exonuclease PCR data with conventional PCR results. bcl-2/JH fusion sequences were detected by exonuclease-based PCR in 24 of 25 cases shown to be bcl-2 rearranged by conventional PCR. Fusion sequences were not detected in patients who were negative by conventional PCR. The overall concordance between the two assays was 98% (52 of 53 cases concordant positive or negative). In a serial dilution study using t(14;18)-positive cell line DNA, exonuclease-based PCR detected fusion sequences at DNA concentrations of 5 pg, equivalent to 0.6 to 0.8 genomes per reaction. Thus, this study demonstrated that exonuclease-based PCR for t(14;18) is both specific and highly sensitive. The elimination of the post-PCR amplicon detection steps and the ability to quantitate the input target DNA sequences make this assay ideal for routine diagnostics and monitoring minimal residual disease.

Chromosomes, Human, Pair 14↗

Genomic DNA amplification and the detection of t(2;5)(p23;q35) in lymphoid neoplasms.

Anaplastic large cell lymphoma (ALCL) is an intermediate grade Non-Hodgkin's lymphoma (NHL) characterized by the frequent presence of the t(2;5)(p23;q35). This translocation fuses the nucleophosmin (NPM) gene on chromosome 5q35 to a protein kinase gene (Anaplastic Lymphoma Kinase, ALK) on chromosome 2p23. In order to determine the frequency of t(2;5) we used a DNA polymerase chain reaction (PCR) amplification using genomic DNA, 5'-primers derived from the NPM gene, and 3'-primers derived from the ALK gene. The presence of amplifiable DNA in the samples was established with PCR and oligonucleotide primers designed to amplify a 3,016 bp fragment from the beta-globin locus. The t(2;5) PCR assay was established using DNA isolated from three t(2;5)-positive ALCL cell lines. Its ability to amplify genomic DNA prepared for routine molecular diagnostic use was validated using archival DNA from four ALCL tumors known to be t(2;5)-positive. Its sensitivity was established by serially diluting t(2;5)-positive DNA in normal DNA: amplicons were generated in 100% of reactions diluted 10(4)-fold (6-8 cells per tube) and in 30% of those diluted 10(5)-fold (0.6-0.8 cells per tube.) We subsequently analyzed archival genomic DNA extracted from 38 ALCL, 77 NHLs, 37 Hodgkin's lymphomas, and 9 lymphomatoid papuloses. The t(2;5) was detected in 6 ALCLs (16%, 95% confidence intervals 6%-31%), but not in any other lymphoma, or in lymphomatoid papulosis. By using the published sequence of the fourth NPM intron that is involved in t(2;5) and by sequencing the individual tumor amplicons and also the normal ALK intron that is involved in t(2;5), we established that all breakpoints involve the same introns in the ALK and NPM loci. Detailed analysis demonstrated that each translocation generates a unique breakpoint sequence, and suggested that sequence homology between the ALK and NPM intron sequences may be involved in the translocation. We conclude that genomic DNA-PCR is useful for the detection of t(2;5) that in our patient population is restricted to ALCL and is not detectable in other NHL, Hodgkin's disease, or lymphomatoid papulosis. More work is needed to determine the prognostic significance of t(2;5), and to establish the utility of the genomic DNA PCR in monitoring minimal residual disease.

Anaplastic Lymphoma Kinase↗

Dying with dignity.

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Attitude to Death↗

Mapping of genomic t(2;5)(p23;q35) break points in patients with anaplastic large cell lymphoma by sequencing long-range PCR products.

The t(2;5) (p23;q35) that is frequently detected in anaplastic large cell lymphoma (ALCL) fuses the nucleophosmin (NPM) gene on chromosome 5 to a novel tyrosine kinase gene designated anaplastic lymphoma kinase (ALK) on chromosome 2. The fusion of NPM and ALK genes results in the production of chimeric transcripts containing NPM amino-terminal sequences fused to the ALK carboxy-terminal catalytic domain. Because fusion transcripts and proteins in almost all t(2;5)-positive cell lines and tumors are identical, it is likely that the chromosomal breaks involve the same introns of NPM and ALK genes. We have previously developed a long-range genomic DNA-PCR assay to amplify the genomic NPM-ALK break points. Using high-molecular-weight DNA extracted from 2 ALCL cell lines and from 9 primary ALCLs known to be t(2;5)-positive, we have demonstrated that all 11 amplicons were of different sizes, suggesting that the t(2;5) break points were unique and involved the same introns on both chromosomes. We decided to confirm this and map the t(2;5) break points by genomic DNA sequencing. Using the same long-range DNA-PCR technique, primers from the ALK locus, and normal genomic DNA, we sequenced the ALK intron involved in t(2;5). We subsequently sequenced all 11 amplicons from t(2;5)-positive ALCL cell lines and tumors. Comparison of the sequences derived from ALCL amplicons with the published sequences of intron 4 from the NPM locus (910 bp) and with the newly sequenced intron from the ALK locus (1935 bp) accurately mapped all break points and demonstrated that their nucleotide sequences were unique. We conclude that the genomic t(2;5) break points can be easily mapped by sequencing the amplicons generated from genomic DNA with long-range PCR and that they are unique for each patient. The sequences of the break points and of the newly identified ALK intron may be useful in the construction of patient-specific primers for monitoring and determination of the clinical relevance of minimal residual disease.

Adolescent↗

The application of fluorescence-based PCR and PCR-SSCP to monitor the clonal relationship of cells bearing the t(14;18)(q32;q21) in sequential biopsy specimens from patients with follicle center cell lymphoma.

This study evaluates the utility of fluorescence-based polymerase chain reaction (PCR) and PCR-SSCP methodologies to monitor the clonal relatedness of cells with bcl-2 major break point region (mbr)/JR fusion sequences in sequential samples from patients with follicular lymphoma (FL). Fluorescence-tagged PCR products from 2-4 sequential samples from seven FL patients were resolved in acrylamide gels and analyzed on an Applied Biosystems' automated DNA sequencer equipped with Genescan software. The amplicons were sequenced directly using automated DNA sequencing to obtain the precise amplicon size and base sequence. Fluorescence-based PCR-single-strand conformation polymorphism (SSCP) analysis performed to distinguish amplicons of similar size but of different base sequence. Amplification products differing by as few as 5 bp resolved clearly under fluorescent PCR assay conditions making possible by visual inspection alone the distinction of two products that otherwise appeared to be of similar size by conventional gel electrophoretic methods. The size of the amplicons as determined by Genescan software correlated exactly with the sizes generated by sequence analysis confirming the precision and accuracy of the fluorescent PCR assay. Under nondenaturing conditions, the mobility profiles of the amplicons from sequential samples with identical base sequence remained indistinguishable, whereas amplicons of similar size but of dissimilar base sequence from different patients exhibited distinct migration patterns. Thus, this study demonstrates that a combination of fluorescent PCR and PCR-SSCP assays for the detection of the t(14;18) provides an accurate measure of clonal relationship based on molecular size and sequence similarities without involving radiolabeling and sequencing strategies. Furthermore, the demonstrated preservation of junctional sequences across sequential biopsy specimens validates the use of PCR in the monitoring of minimal residual disease and eliminates concern about the detection of secondary, non-tumor-related translocations.

Chromosomes, Human, Pair 14↗

Long-range amplification of genomic DNA detects the t(2;5)(p23;q35) in anaplastic large-cell lymphoma, but not in other non-Hodgkin's lymphomas, Hodgkin's disease, or lymphomatoid papulosis.

DESIGN: Determine the frequency of t(2;5)(p23;q35) in anaplastic large-cell lymphoma (ALCL), non-Hodgkin's lymphoma (NHL), Hodgkin's disease (HD), and lymphomatoid papulosis (LP). PATIENTS AND METHODS: The t(2;5) was detected with a long-range nested polymerase chain reaction (PCR) using 0.5 microgram of DNA (60,000-80,000 cells), 5'-primers from the NPM gene, 3'-primers from the ALK gene, agarose electrophoresis, hybridization, and autoradiography. Patients were evaluable if a 3016 base pair amplicon could be generated from tumor DNA with beta-globin primers. RESULTS: Amplicons were detected by PCR of genomic DNA from three ALCL cell lines and four primary ALCLs known to t(2;5) positive. DNA from t(2;5)-positive cell lines diluted 10(4)-fold or 10(5)-fold generated amplicons in 100% or 20% of reactions, respectively. Archival tumor DNA from 144 patients was amplifiable by beta-globin amplicons in 126 (88%) who are considered evaluable for this study. Twenty-two had ALCL, 69 other NHLs, 30 HD, and five LP. Genomic DNA PCR detected the t(2;5) in 5 of 22 with ALCL (23%, 95% confidence intervals [95% CI] 8%-45%) but not in those with NHLs, HD, or LP. Among ALCLs the t(2;5) was confined to 5 of 20 with nodal presentations (25%, 95% CI 9%-49%), among whom it was seen in 5 of 15 with T-cell or null-cell phenotype (33%, 95% CI 12%-62%), in 4 of 11 with age < 40 years (36%, 95% CI 11%-69%), and in 4 of 9 with nodal presentations, T-cell or null-cell phenotype, and age < 40 years (44%, 95% CI 14%-79%). Amplicon sizes were different between cell lines and patients, reflecting unique genomic DNA breakpoints, as confirmed by DNA sequencing, and served as an internal control against specimen cross-contamination in the laboratory. CONCLUSIONS: Long-range PCR of genomic DNA detects t(2;5) only in ALCL, but not in other NHLs, HD, or LP. Long-range PCR may be useful in establishing diagnosis, determining prognosis, and monitoring minimal residual disease in ALCL.

Adolescent↗

Amplification of genomic DNA demonstrates the presence of the t(2;5) (p23;q35) in anaplastic large cell lymphoma, but not in other non-Hodgkin's lymphomas, Hodgkin's disease, or lymphomatoid papulosis.

Anaplastic large cell lymphoma (ALCL) is a distinct clinicopathologic variant of intermediate grade non-Hodgkin's lymphomas (NHL) composed of large pleomorphic cells that usually express the CD30 antigen and interleukin (IL)-2 receptors, and is characterized by frequent cutaneous and extranodal involvement. With variable frequency ALCL bear the t(2;5)(p23;q35) chromosomal translocation that fuses the nucleophosmin (NPM) gene on chromosome 5q35 to a novel protein kinase gene, Anaplastic Lymphoma Kinase (ALK), on chromosome 2p23. We determined the frequency of this translocation with a novel DNA polymerase chain reaction (PCR) technique using 0.5 microgram of genomic DNA, 5'-primers derived from the NPM gene and 3'-primers derived from the ALK gene and hybridization with internal probes. The presence of amplifiable DNA in the samples was tested with the inclusion in the PCR reaction of oligonucleotide primers designed to amplify a 3016-bp fragment from the beta-globin locus. NMP-ALK fusion amplicons were detected using DNA isolated either from all three ALCL cell lines tested, or from all four primary ALCL tumors known to contain the t(2;5)(p23;q35) translocation. Nested amplicons were detected by hybridization in 100% of specimens diluted 10(4)-fold and in 20% of those diluted 10(5)-fold. We subsequently examined archival genomic DNA from 20 patients with ALCL, 39 with diffuse large cell, 2 with mantle cell, 20 with peripheral T cell, 13 with low-grade NHL, 31 with Hodgkin's disease (HD), and 6 with lymphomatoid papulosis. Fusion of the NPM and ALK genes was detected in three of 18 patients with ALCL who had amplifiable DNA (17%, 95% confidence intervals 4% to 41%), but not in any patients with other NHL, HD, or lymphomatoid papulosis. The amplicon sizes were different in all cell lines and patients reflecting unique genomic DNA breakpoints. We conclude that with genomic DNA-PCR the rearrangement of the NPM and ALK loci is restricted to patients with ALCL. Further studies are needed to determine the prognostic significance of the NPM-ALK rearrangement, to determine whether its detection can aid in the differential diagnosis between ALCL. Hodgkin's disease, and lymphomatoid papulosis, and to establish the usefulness of the genomic DNA PCR in the monitoring of minimal residual disease in those patients whose tumors bear the t(2;5).

Adolescent↗

Postoperative management of cataract surgery patients by ophthalmologists and optometrists.

OBJECTIVES: To assess the frequency and content of post-operative examinations by ophthalmologists and optometrists for cataract surgery patients without operative complications and to assess the referral patterns of optometrists when complications are identified. DESIGN: In 1992 we conducted a survey of randomly selected members of the American Academy of Ophthalmology and American Optometric Association. Responses were obtained from 538 (82%) of 655 eligible ophthalmologists and 130 (84%) of 154 eligible optometrists. RESULTS: Eighty-eight percent of responding ophthalmologists reported that patients had 4 or more visits within 4 months after surgery, 97% of ophthalmologists performed the first postoperative examination on their cataract surgery patients, and 60% of ophthalmologists reported that no other eye professional saw their patients postoperatively. Forty-six percent of responding optometrists participated in postoperative care of cataract surgery patients, and usually performed their first postoperative examination 7 days after surgery; 78% of these optometrists reported that they saw patients 3 or more times after surgery. Postoperatively, 83% of ophthalmologists and 75% of optometrists usually performed at least 1 dilated fundus examination, 87% of ophthalmologists and 47% of optometrists performed 4 or more slit-lamp examinations, 74% of ophthalmologists and 42% of optometrists performed 4 or more tonometry tests, and 83% of both groups performed 2 or more refractions. More than 80% of responding optometrists involved in postoperative care of cataract surgery patients immediately refer a patient to an ophthalmologist if there is evidence of acute glaucoma or an unexplained decrease in vision in the eye that was operated on. For less urgent complications, most optometrists promptly make a referral to an ophthalmologist. CONCLUSIONS: In 1992, a small percentage of ophthalmologists and optometrists were performing fewer follow-up examinations and tests for cataract patients than recommended by the American Academy of Ophthalmology. Not all optometrists immediately refer to an ophthalmologist any acute complication that they identify postoperatively.

Adult↗

T-cell-rich B large-cell lymphoma simulating lymphocyte-rich Hodgkin's disease.

Immunophenotypic analysis of 50 cases fulfilling the histologic criteria for mixed cellularity Hodgkin's disease disclosed nine cases with a B-cell, non-Hodgkin's phenotype (CD20+, CD15-, CD30-, EMA-). The cases were characterized by a diffuse small lymphocytic milieu, interspersed atypical large cells including classic Reed-Sternberg cells, and infrequent plasma cells, eosinophils, and L&H cells. The male:female ratio was 7:2 (aged 22-65 years, median 39 years). Three patients were Ann Arbor stage II, two stage III, and four stage IV. The patients presented with generalized lymphadenopathy (four), mesenteric lymph node involvement (two), splenomegaly (four), and bone marrow involvement (three). Four patients were treated with standard Hodgkin's disease protocols. Two attained a complete response and two a partial response; all relapsed and died. Four of five patients treated for large-cell lymphoma achieved a complete response and are currently alive without evidence of disease. The one patient with an initial partial response relapsed and died. We conclude that immunophenotypic analysis is essential in cases of histologic mixed cellularity Hodgkin's disease, especially in those with lymphocyte-rich morphology. Cases with a B-cell phenotype should be diagnosed and treated as T-cell-rich B large-cell lymphoma.

Adult↗

Elimination of both E1 and E2 from adenovirus vectors further improves prospects for in vivo human gene therapy.

A novel recombinant adenovirus vector, Av3nBg, was constructed with deletions in adenovirus E1, E2a, and E3 regions and expressing a beta-galactosidase reporter gene. Av3nBg can be propagated at a high titer in a corresponding A549-derived cell line, AE1-2a, which contains the adenovirus E1 and E2a region genes inducibly expressed from separate glucocorticoid-responsive promoters. Av3nBg demonstrated gene transfer and expression comparable to that of Av1nBg, a first-generation adenovirus vector with deletions in E1 and E3. Several lines of evidence suggest that this vector is significantly more attenuated than E1 and E3 deletion vectors. Metabolic DNA labeling studies showed no detectable de novo vector DNA synthesis or accumulation, and metabolic protein labeling demonstrated no detectable de novo hexon protein synthesis for Av3nBg in naive A549 cells even at a multiplicity of infection of up to 3,000 PFU per cell. Additionally, naive A549 cells infected by Av3nBg did not accumulate infectious virions. In contrast, both Av1nBg and Av2Lu vectors showed DNA replication and hexon protein synthesis at multiplicities of infection of 500 PFU per cell. Av2Lu has a deletion in E1 and also carries a temperature-sensitive mutation in E2a. Thus, molecular characterization has demonstrated that the Av3nBg vector is improved with respect to the potential for vector DNA replication and hexon protein expression compared with both first-generation (Av1nBg) and second-generation (Av2Lu) adenoviral vectors. These observations may have important implications for potential use of adenovirus vectors in human gene therapy.

Adenoviridae↗

Enzymology of indole alkaloid biosynthesis in Catharanthus roseus.

Indole alkaloids in Catharanthus roseus have been in focus because of their medicinal value. These alkaloids consist of an indole moiety provided by tryptamine and a terpenoid portion provided by the secologanin. The most important catharanthus alkaloids are vinblastine (VLB), vincristine (VCR) and ajmalicine. VLB and VCR are clinically useful anticancer agents whereas ajmalicine is used for the treatment of circulatory diseases. VCR and VLB are the most expensive because of their low abundance in the plant, and are formed by the coupling of monomeric indole alkaloids vindoline and catharanthine, catalysed by peroxidases. The pathway that lead to monomeric indole alkaloids involves more than 20 enzymes of which 16 enzymes have been isolated and characterized biochemically, and only three at the molecular level. The present state of knowledge on enzymes and genes involved in indole alkaloid biosynthesis and various aspects of their regulation has been discussed.

Alkaloids↗

Variation in ophthalmic testing prior to cataract surgery. Results of a national survey of optometrists. Cataract Patient Outcome Research Team.

OBJECTIVE: To assess variation in optometrists' use of ophthalmic tests in the evaluation of patients being considered for cataract surgery who have no history of other eye disease. DESIGN/PARTICIPANTS: National survey of a systematic sample of practicing members of the American Optometric Association (St Louis, Mo), who had referred at least one patient to an ophthalmologist for consideration of cataract surgery in 1991. RESULTS: Ninety-two of 130 eligible responding optometrists reported that they routinely performed preoperative testing on patients being considered for cataract surgery. Of these 92 optometrists, 91 (99%) frequently or always performed refraction, and 82 (89%) frequently or always performed a dilated fundus examination in their evaluation of patients being considered for cataract surgery who had no history of other eye disease. None of these 92 optometrists reported using B-scan ultrasonography or electroretinograms frequently or always, and few used A-scan ultrasonography or visual evoked responses frequently or always. A substantial percentage frequently or always used formal visual field testing (47%), formal color vision testing (40%), fundus photography (24%), potential acuity measurement (25%), glare testing (23%), contrast sensitivity testing (19%), and specular microscopy (14%), while 11% to 81% of optometrists never performed these tests on such patients. More recent graduation from optometry school was associated with a decreased frequency of use of potential acuity measurement and contrast sensitivity testing and with an increased use of dilated fundus examinations. CONCLUSION: There is a substantial variation in optometrists self-reported use of a number of ophthalmic tests in the preoperative evaluation of patients being considered for cataract surgery who have no history of other eye disease.

Adult↗

Cataract surgical techniques. Preferences and underlying beliefs.

To characterize the intraoperative procedures employed by cataract surgeons in the United States and the beliefs underlying the practices, a standardized questionnaire was sent to a systematic random sample of members of the American Academy of Ophthalmology in 1992. Of 667 surveyed ophthalmologists, 550 completed the questionnaire (response rate, 82.5%). Phacoemulsification was used for more than 75% of routine cataract surgery by 46% of respondents, whereas standard extracapsular surgery was used for more than 75% of routine cataract surgery by 41% of respondents. Preferential use of phacoemulsification was independently associated with more recent graduation from medical school and higher reported annual surgical volume. Continuous tear capsulotomy was employed by 52% of ophthalmologists. Preference for this technique was independently associated with both the use of phacoemulsification and higher annual surgical volume. Seventy-one percent of respondents used retrobulbar anesthesia, whereas 28% used peribulbar anesthesia. Use of peribulbar anesthesia was independently associated with both greater surgical volume and performance of surgery in an ambulatory surgical center. Beliefs regarding comparative safety and effectiveness were reported to influence surgeons' preferences strongly among all of the competing techniques studied. Those performing phacoemulsification, in comparison with those performing extracapsular cataract extraction, reported that the expectation of reduced astigmatism and shorter recovery time strongly influenced their choice of procedure. Variation in preferred intraoperative techniques is substantial for cataract surgery and the beliefs that underlie the preferences. Such variation highlights the need to determine which techniques maximize patient outcomes and are most cost-effective.

Adult↗

Do ophthalmologists, anesthesiologists, and internists agree about preoperative testing in healthy patients undergoing cataract surgery?

To assess variation in reported use of preoperative medical tests in patients undergoing cataract surgery and to identify factors that influence test use by different physician groups we performed a national survey of ophthalmologists, anesthesiologists, and internists. Participants included randomly selected members of American professional societies who provided care to one or more patients undergoing cataract surgery in 1991. Responses were obtained from 538 (82%) of 655 eligible ophthalmologists, 109 (76%) of 143 anesthesiologists, and 54 (44%) of 122 internists. Fifty percent of ophthalmologists, 40% of internists, and 33% of anesthesiologists frequently or always obtained a chest x-ray film, while 20% of ophthalmologists, 27% of internists, and 37% of anesthesiologists never obtained a chest x-ray film for patients being considered for cataract surgery who had no history of major medical problems (P < .01 for differences between ophthalmologists and the other groups). Similarly, 70% to 90% of ophthalmologists, 73% to 79% of internists, and 41% to 79% of anesthesiologists frequently or always obtained a complete blood cell count, electrolyte panel, and electrocardiogram, while 4% to 11% of ophthalmologists, 13% to 17% of internists, and 9% to 28% of anesthesiologists never obtained these tests for such patients. Many respondents (32% to 80%) believed tests were unnecessary but cited multiple reasons for obtaining tests (eg, medicolegal concerns and institutional requirements). Many physicians in each group viewed preoperative evaluations as screening opportunities or believed that one of the other two types of physicians "required" tests. We conclude that marked variation exists within and across physician specialties in the use and rationale for use of medical tests in patients undergoing cataract surgery.

Anesthesiology↗

Polymerase chain reaction detection of the t(11;14) translocation involving the bcl-1 major translocation cluster in mantle cell lymphoma.

The utility of polymerase-mediated assays in the detection of the t(11;14) involving the bcl-1 major translocation cluster (bcl-1 MTC) was evaluated by analyzing DNA from 33 patients with mantle cell lymphoma, 14 patients with other non-Hodgkin's lymphomas, and five patients with reactive lymphoid hyperplasia. The polymerase chain reaction (PCR) assay was performed using a consensus immunoglobin heavy-chain joining region primer in conjunction with a chromosome 11 specific oligonucleotide primer flanking the translocation site. The sensitivity and specificity of the assay were confirmed by correlation of the (PCR) assay data with restriction analysis. Rearrangements at the bcl-1 MTC were detected in 13 (39%) of 33 cases of mantle cell lymphoma by PCR and in 13 (48%) of 27 cases by restriction analysis. Amplicons were detectable by PCR in 85% (11 of 13) of the cases shown to be bcl-1 rearranged by restriction analysis. Failure to detect amplification products in DNA samples from non-mantle cell lymphomas and reactive follicular hyperplasia further confirmed the specificity of the assay. Sequential hybridization of the PCR products with oligonucleotide probes 3' to the bcl-1 MTC primer revealed that the breakpoints in the bcl-1 MTC were clustered around an Sst I restriction site over a range of 170 base pairs. The study demonstrates that PCR-mediated assay for the detection of the t(11;14) at the bcl-1 MTC is specific and sensitive and can be used as an adjunct to restriction analysis in routine diagnostics.

Base Sequence↗