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"Reconstituted" [alpha]-thalassemia genomic samples as positive controls for the molecular diagnostic laboratory.

We and others recently described strategies for multiplex-PCR analysis of deletional determinants of [alpha]-thalassemia (1-3), culminating in the development of a single-tube assay for simultaneous screening of seven common deletions (4). Since then, several molecular diagnostic laboratories wanting to set up the test have requested DNA samples carrying these deletions for use as validation and positive controls. These requests have led to a critical shortage of our limited stocks of genomic samples, especially those with the rarer deletions, something that has caused our inability to fulfill all requests. The ideal solution to limited genomic DNA is to establish immortal lymphoblastoid cell lines from peripheral-blood leukocytes of patients by Epstein--Barr virus transformation (5, 6). To do so, however, requires that patients be contacted again to provide renewed consent and a fresh aliquot of blood for transformation, something that may be inconvenient or impractical for third-party referral laboratories to implement.

We have devised an alternative strategy for creating a renewable resource of positive control samples of known [alpha]-thalassemia genotype, derived from existing patient DNA samples. Briefly, genomic DNA of known [alpha]-thalassemia genotype was initially used as a template to amplify each deletion junction fragment individually by PCR. The relevant primer pairs and thermo-cycling conditions were as described previously (4). Junction fragments of seven [alpha]-thalassemia deletions were PCR-amplified separately from patient DNA, gel-purified with the GFXTM reagent set (Amersham Pharmacia Biotech), and ligated to a pBluescript (Stratagene) T-vector (Fig. 1A). Each 5-[micro]L ligation reaction contained 15 ng of T-vector and an amount of PCR product, which led to a final vector-to-insert molar ratio of 1:3, and 1 U of T4 DNA ligase in 1 x supplied ligation buffer (Fermentas). Ligation reactions were incubated at 16 [degrees]C for 16 h, and products were transformed into DH5[alpha][TM] competent cells (Life Technologies). Recombinant clones containing each junction fragment were identified by PCR amplification with appropriate insert- and vector-specific primers, and verified recombinant plasmid DNA was isolated by standard techniques (7).

To generate a reconstituted genomic DNA sample heterozygous for an [alpha]-thalassemia deletion, recombinant plasmid DNA containing the relevant deletion junction fragment was mixed with genomic DNA extracted from either a lymphoblastoid cell line or peripheral-blood lymphocytes that were previously determined to be homozygous normal (nondeleted) at the [alpha]-globin locus. Multiplex-PCR amplifications were performed as described (4), and one-fifth of each product was resolved across a 1% agarose, 1x Tris-borate-EDTA gel at 15 V/cm for 1 h. Beginning with equimolar mixes of haploid genome equivalents and supercoiled plasmid constructs, we empirically adjusted the molar ratios such that the multiplex-PCR results for each of the reconstituted heterozygous positive-control samples closely resembled those obtained from actual heterozygous patient samples. We further readjusted the ratios of plasmid constructs to normal genomic DNA such that the signal intensities of the amplified deletion junction fragments relative to the normal [alpha]2 fragment were weaker coming from reconstituted positive controls than from actual heterozygous patients (Table 1). The higher stringency for detection of deletion junction fragments from reconstituted positive controls compared with actual samples was intended to ensure that a negative diagnostic test result was real when positive amplification was observed from reconstituted positive controls. Conversely, false-negative diagnostic results that could occur because of suboptimal PCR conditions would not be expected to be missed because the reconstituted positive controls would be the first to fail under the same conditions. As shown in Fig. 1B, the reconstituted samples yielded amplification results resembling those from actual heterozygous DNA samples, but with weaker ratios of deletion junction fragment to normal [alpha]2 fragment signal intensities.

[FIGURE 1 OMITTED]

Interestingly, a molar excess of plasmid construct to haploid genome equivalents is still required in each reconstituted positive control (Table 1), although actual heterozygous patient samples contained an equal ratio of normal and deletion alleles. This disparity in template requirement could be attributable to lower amplification efficiencies from supercoiled DNA resulting from stearic hindrances and/or rapid complementary-strand reannealing during the primer-annealing step. This hypothesis is supported by our observation of stronger signal intensities of the deletion junction fragments when plasmid constructs were linearized before mixing with genomic DNA (data not shown). Although prelinearization of the plasmid constructs enables molar ratios to be closer to parity, supercoiled plasmids may be the preferred form for long-term storage as mixtures with genomic DNA because they are less susceptible than linearized plasmids to trace amounts of nucleases.

Another approach to reconstituting positive-control samples, an approach we initially used before cloning all the junction fragments, is to mix the isolated PCR-amplified deletion junction fragments directly with genomic DNA. Although this method works equally well and obviates the need for cloning, all the other optimization steps are still necessary. Furthermore, the amount of plasmid DNA that can be obtained from a single bacterial maxi-preparation far exceeds the amount of the deletion junction fragment that can be obtained by PCR, yielding sufficient stable DNA to generate innumerable positive controls indefinitely without the need for periodic re-PCR and fragment isolation.

We also assessed the feasibility of using as positive controls the diluted multiplex-PCR products of patients who were positive for deletions. In a test using multiplex-PCR product from a -[[alpha].sup.3.7] deletion carrier diluted over more than three different orders of magnitude, however, we were unable to generate results anywhere resembling the original multiplex-PCR result, with only the normal [alpha]2 fragment amplifying strongly (data not shown). This observation was not surprising considering that the relative ratios of -[[alpha].sup.3.7], [alpha]2, and LIS1 copies in a genomic-DNA sample are unlikely to be maintained in the amplified product.

Furthermore, we also assessed the feasibility of generating a single-tube "multipositive control" by mixing all seven plasmid constructs together with normal genomic DNA, in the amounts indicated in Table 1. All nine fragments (seven deletion junction fragments plus the [alpha]2 and LIS1 control fragments) amplified successfully from the reconstituted sample (Fig. 1C). The signal intensities of the various deletion junction fragments relative to the normal [alpha]2 fragment were also comparable with the corresponding relative intensities observed in the individually reconstituted positive controls. Use of a multi-positive control sample such as this would reduce the number of positive control reactions necessary in a diagnostic test by sevenfold. It remains to be seen whether such a reconstituted genomic sample containing multiple deletion alleles will find wide acceptance as a sufficiently "authentic" positive control for use in a diagnostic-test setting.

In summary, we have successfully used a strategy to create "reconstituted" genomic DNA samples heterozygous for each of the seven deletions screened for in our multiplex-PCR assay, and these renewable reagents are now available on request. Genomic DNA samples of known mutational genotype are necessary reagents in the molecular diagnostic laboratory, serving a critical role as positive controls during the testing of clinical samples. Our reconstitution strategy represents a convenient, rapid, and inexpensive alternative to cell immortalization techniques for generating renewable positive controls for [alpha]-thalassemia multiplex-PCR testing and should be generally applicable to other inherited disorders where immortalized cell lines are not readily available.

This work was supported by Grant NMRC/0365/1999 (Singapore) to S.S.C.

References

(1.) Chong SS, Boehm CD, Higgs DR, Cutting GR. Single-tube multiplex-PCR screen for common deletional determinants of [alpha]-thalassemia. Blood 2000; 95:360-2.

(2.) Liu YT, Old JM, Miles K, Fisher CA, Weatherall DJ, Clegg JB. Rapid detection of [alpha]-thalassemia deletions and [alpha]-globin gene triplication by multiplex PCRs. Br J Haematol 2000;108:295-9.

(3.) Chong SS, Boehm CD, Cutting GR, Higgs DR. Simplified multiplex-PCR diagnosis of common Southeast Asian deletional determinants of [alpha]-thalassemia. Clin Chem 2000;46:1692-5.

(4.) Tan AS, Quah TC, Low PS, Chong SS. A rapid and reliable 7-deletion multiplex polymerase chain reaction assay for [alpha]-thalassemia. Blood 2001;98:250-1.

(5.) Miller G, Lisco H, Kohn HI, Stitt D, Enders JF. Establishment of cell lines from normal adult human blood leukocytes by exposure to Epstein-Barr virus and neutralization by human sera with Epstein-Barr virus antibody. Proc Soc Exp Biol Med 1971;137:1459-65.

(6.) Nilsson K, Klein G, Henle W, Henle G. The establishment of lymphoblastoid lines from adult and fetal human lymphoid tissue and its dependence on EBV. Int J Cancer 1971;8:443-50.

(7.) Sambrook J, Fritsch EF, Maniatis T, eds. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989;1:1.1-1.110.

Wen Wang, [1] Arnold S-C. Tan, [1] and Samuel S. Chong [1,2,3] *

[1] Departments of Pediatrics and Obstetrics and Gynecology, National University of Singapore, Singapore 119074, Singapore;

[2] Molecular Diagnosis Center, Department of Laboratory Medicine, National University Hospital, Singapore 119074, Singapore;

[3] Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, MD 21205;

* address correspondence to this author at: Department of Pediatrics, National University of Singapore, Level 4, Main Building, National University Hospital, 5 Lower Kent Ridge Rd., Singapore 119074, Singapore; fax 65-6779-7486, e-mail paecs@nus.edu.sg)
Table 1. Generation of heterozygous [alpha]thalassemia genomic
samples: optimization of mixtures of normal genomic DNA with plasmid
constructs carrying deletion junction fragments.

Deletion Junction Construct Molecular weight
 fragment, size, bp of construct
 bp

~ 2029 4990 3.29 x [10.sup.6]
[-[alpha].sup.4.2] 1628 4589 3.03 x [10.sup.6]
[--.sup.SEA] 1249 4310 2.84 x [10.sup.6]
[--.sup.THAI] 1153 4114 2.72 x [10.sup.6]
[-[alpha].sup.20.5] 1007 3968 2.62 x [10.sup.6]
[--.sup.MED] 807 3768 2.48 x [10.sup.6]
[--.sup.FIL] 546 3507 2.31 x [10.sup.6]

Deletion Construct Molar ratio
 added per (deletion:
 100 ng of [[alpha]2.
 gDNA, sup.c])
 (a,b) pg

~ 0.566 ~3.5:1
[-[alpha].sup.4.2] 0.459 ~3:1
[--.sup.SEA] 0.714 ~5:1
[--.sup.THAI] 1.176 ~8.5:1
[-[alpha].sup.20.5] 0.227 ~1.5:1
[--.sup.MED] 0.271 ~2:1
[--.sup.FIL] 0.75 ~6.5:1

(a) One hundred nanograms of human genomic DNA contain ~3 X [10.sup.4]
haploid genome-equivalents. Normal human genomic DNA contains 1 copy
of the [alpha]2 globin gene per haploid genome.
(b) gDNA, genomic DNA.
(c) Ratio of [alpha]2 globin gene copies to deletion junction copies
in reconstituted genomic samples necessary to achieve results similar
to actual heterozygous DNA samples.
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Title Annotation:Technical Briefs
Author:Wang, Wen; Tan, Arnold S-C.; Chong, Samuel S.
Publication:Clinical Chemistry
Date:Jun 1, 2002
Words:1737
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