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Microtransponder-based multiplex assay for genotyping cystic fibrosis.

Cystic fibrosis (CF) is caused by one or more mutations in the gene encoding for the CF transmembrane conductance regulator (CFTR) protein. CF occurs when both copies of the CFTR gene function abnormally, and one functional copy is sufficient to prevent the disease. In the Caucasian population, CF is inherited with a frequency of 1:3300, making it the most lethal inherited disease of childhood, but carrier frequency and incidence of CF vary with race and ethnic group (1). A single mutation causing loss of the phenylalanine residue at position 508 ([Delta]F508) accounts for nearly 70% of all mutations observed in Caucasians with CF, but more than 1000 other mutations of the CFTR gene have been reported in all races and ethnic groups. In May 2001, the American College of Medical Genetics published a recommended panel of 25 mutations and 6 polymorphisms for population-based CF screening (2).

We describe a new platform for performing a multiplexed genotyping assay based on radio frequency (RF) microtransponders (MTPs) and provide a working example, the CF assay. CF mutations tested and the rationale are provided in Table 1 and Supplemental Data (see the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol53/issue7. We designed this assay to be ethnic-group specific, thus simplifying the design and potentially reducing costs. The mutations are grouped into 4 panels: core, Caucasian, Hispanic, and African-American. Because the assay is ethnic specific, efficiency is increased, with fewer DNA probes and reagents needed. A patient would be tested with 2 panels, the core panel and one ethnic panel; more than one ethnic panel might be used for patients with complex ethnic backgrounds.

The key element of the assay method is the MTP (Fig. 1A), a monolithic 500 [micro]m x 500 [micro]m integrated circuit chip that can transmit its identification code by RF. Each chip consists of photocells, read-only memory (ROM), transmit logic circuitry, and an antenna loop. Visible light, typically red or green, is pulsed at 1.5 MHz to provide power and a stable clocking signal for the logic circuitry. The circuitry accesses the contents of ROM (the ID value) and modulates the current through the antenna in correspondence with the ID value. The resulting variable magnetic field in the vicinity of the MTP can then be measured with a nearby receiving coil and decoded to provide the ID value, which identifies the oligonucleotide immobilized on the MTP. The current MTP design uses 10 bits to encode the ID value, allowing 1024 unique values; however, the ROM contains an additional 40 unused bits, so the MTPs could be manufactured to have as many as [2.sup.50] (~[10.sup.15]) unique ID values. Before use, the MTPs are coated with a polymer that places both hydroxy and amino groups on the surface of the chip. Such derivatized MTPs are subjected to oligonucleotide synthesis.

In preparation for the flow reader analysis, the MTPs are suspended in a liquid medium that prevents sedimentation of MTPs but allows flow characteristics comparable to water when sheared. The suspension is repeatedly passed through a narrow channel of the instrument, where the ID values are read and fluorescence measurements are made. The flow system is designed to support a transfer rate of up to 1000 MTPs/s. The time needed to read the ID can be as short as 300 [micro]s and the time to read fluorescence 1-2 ms. Multiple forward and reverse passes, typically 50 total passes, of the MTPs through the flow channel are required to obtain enough data for analysis. The instrument in the present study, Tsunami IV, uses a 532-run, 300-mW laser for both RF identification and fluorescence at a single location on one side of the flow channel. We used custom software named Retro for data analysis. Both the flow reader and MTPs are described in more detail in a recent report (3).

The principle for detecting mutations is allele-specific primer extension (ASPE) (4,5) on the PCR-amplified CFTR DNA; the schematic of the assay is shown in Fig. 1B. For each mutation, 2 primers were prepared, one specific to the wild-type allele and another specific to the mutant allele. The sequence differences between the 2 primers are the tag sequence at the 5' end and a single nucleotide at the 3' end (an "anchor" sequence). Thus, for any particular allele, only 1 primer was extended in a reaction with DNA polymerase in the presence of 4 dNTPs. After the ASPE reaction, the target DNA was hybridized to a capture probe, the sequences of which are complementary to tag sequences commonly referred to as universal tags (4, 6-8). The capture probe is a synthetic oligonucleotide (24 nucleotides) covalently bound to the MTP surface. Because the tag sequence was present at the 5' end of allele-specific primers, the capture reaction was very specific. In the ASPE reaction, we used biotin-labeled dCTP in place of dCTP. Thus, the ASPE target typically contained several biotin moieties, which were subsequently visualized as a result of a binding reaction with phycoerythrin-labeled streptavidin (phycoerythrin is a pigment-carrying, highly fluorescent protein from red algae). The fluorescence was then read in the flow reader instrument.

Eighteen pairs of PCR primers were designed using an in-house custom software package, SimuPlex (9). SimuPlex accepts a sequence file comprising all the loci for which PCR primers are to be designed. The SimuPlex then identifies all qualified primers that meet the criteria defined in the parameter file and executes a local BLAST (Basic Local Alignment Search Tool) search that filters out undesired primers. Once the candidates are filtered, SimuPlex creates a "seed" multiplex set and then uses a simulated annealing algorithm to search the surrounding solution space for even better multiplex sets. The melting temperature ([T.sub.m]) and free-energy calculations are based on the most accurate and up-to-date formulas and thermodynamic data sets (10,11). The results of a multiplex PCR reaction for which primers were designed using SimuPlex software is shown in Fig. 1C.

Allele-specific primers containing 24-nt tag sequences were designed using 3 custom programs written in Python: ExtractProbes.py, FindOptProbe.py, and Tags2Probes.py. The tag sequences were at the 5' end of the primers, and allele-specific sequences were at the 3' end. The allelespecific sequences varied in length, but always possessed a [T.sub.m] of 50[degrees]C. For each biallelic single nucleotide polymorphism analyzed by ASPE, 2 allele-specific primers (ASP) were synthesized, with each ASP differing in the tag sequence and in the polymorphic nucleotide contained at its 3' terminus.

We conducted 2 separate series of experiments to validate the performance of the assay. In the internal study, we used 23 standard (Coriell) genomic DNA samples. In addition, we used 32 coded genomic DNA samples in an external study completed in Dr. Dermody's laboratory at the University of Medicine and Dentistry of New Jersey. During the course of this project, >100 CF reagent sets were prepared. Each reagent set consisted of a vial containing derivatized MTPs that compose the mutation panel and the assay file on electronic media. Typically, 3 MTPs were used for each probe to achieve multiple readouts for statistical accuracy.

The results from the internal study (Table 1) indicate that correct calls were 98.8% of all determinations (807 total calls), and false-positive and false-negative calls were 1.1% and 0.12%, respectively. The results from the external study are shown in the Supplemental Data. Correct calls were 95.7% of all determinations (1086 total calls), and false-positive and false-negative calls were 3.9% and 0.36%, respectively. In addition, 27 synthetic 60-nt oligonucleotides were designed to simulate DNA mutations not present in the Coriell DNA samples. Assays performed on the synthetic samples resulted in 100% correct calls of homozygous mutation.

We are generally pleased with the results obtained in both the internal and external testing. The overall percentage of correct calls was high: 98.8% and 95.6%, respectively. Especially encouraging were high fluorescence ratios (wild type-to-mutant oligo probe), approaching 100 in many cases, indicating a high potential of the assay for DNA testing, and in particular CF testing. The wrong calls seem to be clustered for specific mutations, suggesting difficulties with certain oligonucleotides or PCR products. In summary, MTPs were used as solid phase in a CF assay. Although the reported rate of false positives (1%-4%) is higher than in commercially available CF assays [Luminex (12), Roche, and Applera (13)], we are confident that it can be improved, because the biochemical basis of the assay is well understood and the biochemical principle is similar to that implemented in the Luminex CF assay (12). The main advantage of the MTP platform is the large number of ID codes available, currently 1024 but readily expandable. The expansion might be justified if the number of mutations being tested for increases, or if other types of assays require a higher multiplex level.

[FIGURE 1 OMITTED]

Grant funding/support: This work was funded by National Institutes of Health Grant HL074607.

Financial disclosures: The authors associated with PharmaSeq, Inc., have equity interest in the company. J.D. is a consultant to the company.

Acknowledgments: We thank Richard G. Morris and Marvin Schwalb for helpful discussions and encouragement.

Previously published online at DOI: 10.1373/clinchem.2006.081810

References

(1.) Palomaki GE, Fitzsimmons SC, Haddow JE. Clinical sensitivity of prenatal screening for cystic fibrosis via CFTR carrier testing in a United States panethnic population. Genet Med 2004;6:405-14.

(2.) American College of Obstetrics and American College of Medical Genetics. Preconception and prenatal carrier screening for cystic fibrosis: clinical and laboratory guidelines. Washington, DC: American College of Obstetrics and Gynecology, 2001:32pp.

(3.) Mandecki W, Ardelt B, Coradetti T, Davidowitz H, Flint J, Huang Z, et al. Microtransponders, the miniature RFID electronic chips, as platforms for cell growth in cytotoxicity assays. Cytometry A 2006;69:1097-105.

(4.) Ye F, Li MS, Taylor JD, Nguyen Q, Colton HM, Casey WM, et al. Fluorescent microsphere-based readout technology for multiplexed human single nucleotide polymorphism analysis and bacterial identification. Hum Mutat 2001; 17:305-16.

(5.) Taylor JD, Briley D, Nguyen Q, Long K, Lannone MA, Li MS, et al. Flow cytometric platform for high-throughput single nucleotide polymorphism analysis. BioTechniques 2001;30:661-9.

(6.) Tm Bioscience. http://www.tmbioscience.com/tm100universalarray.php (accessed February 2007).

(7.) Wang DG, Fan JB, Siao CJ, Berno A, Young P, Sapolsky R, et al. Large-scale identification, mapping and genotyping of single nucleotide polymorphisms in the human genome. Science 1988;280:1077-82.

(8.) Gerry NP, Witowski NE, Day J, Hammer RP, Barany G, Barany F. Universal DNA microarray method for multiplex detection of low abundance point mutations. J Mol Biol 1999;292:251-62.

(9.) Azaro MA, Lin X, Mandecki W. SimuPlex: primer design software for multiplex PCR. Poster presentation, NIH Small Business Innovation Research Conference, July 2005, Bethesda, MD.

(10.) SantaLucia JJ, Hicks D. The thermodynamics of DNA structural motifs. Annu Rev Biophys Biomol Struct 2004;33:415-40.

(11.) The Santa Lucia Lab. http://ozone3.chem.wayne.edu (accessed February 2007).

(12.) Strom CM, Janeczko R, Quan F, Wang SB, Buller A, McGinniss M, et al. Technical validation of Tm Biosciences Luminex-based multiplex assay for detecting the American College of Medical Genetics recommended cystic fibrosis mutation panel. J Mol Diagn 2006;8:371-5.

(13.) Strom CM, Clark DD, Hantash FM, Rea L, Anderson B, Maul D, et al. Direct visualization of cystic fibrosis transmembrane regulator mutations in the clinical laboratory setting. Clin Chem 2004;50:836-45.

(14.) CDC web site: http://www.cdc.gov/genomics/gtesting/ACCE/FBR/CF/CFCIiVal 19c.htm (accessed February 2007).

(15.) Palomaki GE, Haddow JE, Bradley LA, FitzSimmons SC. Updated assessment of cystic fibrosis mutation frequencies in non-Hispanic Caucasians. Genet Med 2002;4:90-4.

(16.) Kazazian HH. Population variation of common cystic fibrosis mutations. Hum Mutat 1994;4:167-77.

(17.) Tomaiuolo R, Spina M, Castaldo G. Molecular diagnosis of cystic fibrosis: comparison of four analytical procedures. Clin Chem Lab Med 2003;41:26-32.

(18.) Heim RA, Sugarman EA, Allitto BA. Improved detection of cystic fibrosis mutations in the heterogeneous U.S. population using an expanded, pan-ethnic mutation panel. Genet Med 2001;3:168-76.

(19.) Bobadilla JL, Macek M Jr, Fine JP, Farrell PM. Cystic fibrosis: a worldwide analysis of CFTR mutations: correlation with incidence data and application to screening. Hum Mutat 2002;19:575-606.

Xin Lin, [1] James A. Flint, [1] Marco Azaro, [1] Thomas Coradetti, [1] Wesley M. Kopacka, [1] Deanna L. Streck, [2] Zhuying Wang, [1] James Dermody, [2] and Wlodek Mandecki [1] *

[1] PharmaSeq, Inc., Monmouth Junction, NJ;

[2] Department of Microbiology & Molecular Genetics, University of Medicine and Dentistry, New Jersey-New Jersey Medical School, Newark, NJ;

* address correspondence to this author at: PharmaSeq, Inc., 11 Deer Park Drive, Suite 104, Monmouth Junction, NJ 08852; fax 732-355-0102, e-mail mandecki@pharmaseq.com
Table 1. CF genotyping results for the core panel
from internal validation. (a)

Sample Allelic Variant G542X A455E

1 Wild-type DNA . .
2 [Delta]F508/ F508 . .
3 3120 + 1G>A/621 + 1G>T . .
4 R553/[Delta]F508 . .
5 G551D/wild-type . .
6 3659delC/[Delta]F508 . .
7 [Delta]I507/wild-type . .
8 711 + 1G>T/621 + 1G>T . .
9 621 + 1G>T/[Delta]F508 . .
10 G85E/621 + 1G>T . .
11 A455E/[Delta]F508 . m
12 R560T/[Delta]F508 . .
13 N1303K/G1349D . .
14 G542X/G542X M .
15 W1282X/wild-type . .
16 2789 + 5G>A/2789 + 5G>A . .
17 3849 + 10C>T/3849 + 10C>T . .
18 1717-1G>T/wild-type . .
19 R1162X/wild-type . .
20 R347P/G551D . .
21 R334W? . .
22 R117H/[Delta]F508 . .
23 2184delA/[Delta]F508 . .
24 1898 + 1G>A/[Delta]F508 . .

 3 4
Sample Allelic Variant G551D 3659delC

1 Wild-type DNA . .
2 [Delta]F508/ F508 . .
3 3120 + 1G>A/621 + 1G>T . .
4 R553/[Delta]F508 . .
5 G551D/wild-type m .
6 3659delC/[Delta]F508 . m
7 [Delta]I507/wild-type . .
8 711 + 1G>T/621 + 1G>T . .
9 621 + 1G>T/[Delta]F508 . .
10 G85E/621 + 1G>T . .
11 A455E/[Delta]F508 . .
12 R560T/[Delta]F508 . .
13 N1303K/G1349D . .
14 G542X/G542X . .
15 W1282X/wild-type . .
16 2789 + 5G>A/2789 + 5G>A . .
17 3849 + 10C>T/3849 + 10C>T . .
18 1717-1G>T/wild-type . .
19 R1162X/wild-type . .
20 R347P/G551D m .
21 R334W? . .
22 R117H/[Delta]F508 . .
23 2184delA/[Delta]F508 . .
24 1898 + 1G>A/[Delta]F508 . .

 5 6
Sample Allelic Variant R334W 1078delT

1 Wild-type DNA . .
2 [Delta]F508/ F508 . .
3 3120 + 1G>A/621 + 1G>T . .
4 R553/[Delta]F508 . .
5 G551D/wild-type . .
6 3659delC/[Delta]F508 . .
7 [Delta]I507/wild-type . .
8 711 + 1G>T/621 + 1G>T . .
9 621 + 1G>T/[Delta]F508 . .
10 G85E/621 + 1G>T . .
11 A455E/[Delta]F508 . .
12 R560T/[Delta]F508 . .
13 N1303K/G1349D . .
14 G542X/G542X . .
15 W1282X/wild-type . .
16 2789 + 5G>A/2789 + 5G>A . .
17 3849 + 10C>T/3849 + 10C>T . .
18 1717-1G>T/wild-type . .
19 R1162X/wild-type . .
20 R347P/G551D . .
21 R334W? m .
22 R117H/[Delta]F508 . .
23 2184delA/[Delta]F508 . .
24 1898 + 1G>A/[Delta]F508 . .

 7 8
Sample Allelic Variant 1717-1G>A R553X

1 Wild-type DNA . .
2 [Delta]F508/ F508 . .
3 3120 + 1G>A/621 + 1G>T . .
4 R553/[Delta]F508 . m
5 G551D/wild-type . .
6 3659delC/[Delta]F508 . .
7 [Delta]I507/wild-type . .
8 711 + 1G>T/621 + 1G>T . .
9 621 + 1G>T/[Delta]F508 . M*
10 G85E/621 + 1G>T . .
11 A455E/[Delta]F508 . .
12 R560T/[Delta]F508 . .
13 N1303K/G1349D . .
14 G542X/G542X . .
15 W1282X/wild-type . .
16 2789 + 5G>A/2789 + 5G>A . .
17 3849 + 10C>T/3849 + 10C>T . .
18 1717-1G>T/wild-type m .
19 R1162X/wild-type . .
20 R347P/G551D . .
21 R334W? . .
22 R117H/[Delta]F508 . .
23 2184delA/[Delta]F508 . .
24 1898 + 1G>A/[Delta]F508 . .

 9 10
Sample Allelic Variant R560T R1162X

1 Wild-type DNA . .
2 [Delta]F508/ F508 . .
3 3120 + 1G>A/621 + 1G>T . .
4 R553/[Delta]F508 . .
5 G551D/wild-type . .
6 3659delC/[Delta]F508 . .
7 [Delta]I507/wild-type . .
8 711 + 1G>T/621 + 1G>T . .
9 621 + 1G>T/[Delta]F508 . .
10 G85E/621 + 1G>T . .
11 A455E/[Delta]F508 . .
12 R560T/[Delta]F508 m .
13 N1303K/G1349D . .
14 G542X/G542X . .
15 W1282X/wild-type . .
16 2789 + 5G>A/2789 + 5G>A . .
17 3849 + 10C>T/3849 + 10C>T . .
18 1717-1G>T/wild-type . .
19 R1162X/wild-type . m
20 R347P/G551D . .
21 R334W? . .
22 R117H/[Delta]F508 . .
23 2184delA/[Delta]F508 . .
24 1898 + 1G>A/[Delta]F508 . .

 11 12
Sample Allelic Variant R347P 2789 + 5G>A

1 Wild-type DNA . .
2 [Delta]F508/ F508 . .
3 3120 + 1G>A/621 + 1G>T . .
4 R553/[Delta]F508 . .
5 G551D/wild-type . .
6 3659delC/[Delta]F508 . .
7 [Delta]I507/wild-type . .
8 711 + 1G>T/621 + 1G>T . .
9 621 + 1G>T/[Delta]F508 . .
10 G85E/621 + 1G>T . .
11 A455E/[Delta]F508 . .
12 R560T/[Delta]F508 . .
13 N1303K/G1349D . .
14 G542X/G542X . .
15 W1282X/wild-type . .
16 2789 + 5G>A/2789 + 5G>A . M
17 3849 + 10C>T/3849 + 10C>T . .
18 1717-1G>T/wild-type . .
19 R1162X/wild-type . .
20 R347P/G551D m .
21 R334W? . .
22 R117H/[Delta]F508 . .
23 2184delA/[Delta]F508 . .
24 1898 + 1G>A/[Delta]F508 . .

 13 14
Sample Allelic Variant G85E N1303K

1 Wild-type DNA . .
2 [Delta]F508/ F508 . .
3 3120 + 1G>A/621 + 1G>T . .
4 R553/[Delta]F508 . .
5 G551D/wild-type . .
6 3659delC/[Delta]F508 . .
7 [Delta]I507/wild-type . .
8 711 + 1G>T/621 + 1G>T . m*
9 621 + 1G>T/[Delta]F508 . .
10 G85E/621 + 1G>T m .
11 A455E/[Delta]F508 . .
12 R560T/[Delta]F508 . .
13 N1303K/G1349D . m
14 G542X/G542X . m*
15 W1282X/wild-type . .
16 2789 + 5G>A/2789 + 5G>A . .
17 3849 + 10C>T/3849 + 10C>T . .
18 1717-1G>T/wild-type . .
19 R1162X/wild-type . .
20 R347P/G551D . .
21 R334W? . .
22 R117H/[Delta]F508 . .
23 2184delA/[Delta]F508 . .
24 1898 + 1G>A/[Delta]F508 . .

 15 16
Sample Allelic Variant R117H W1282X

1 Wild-type DNA . .
2 [Delta]F508/ F508 . .
3 3120 + 1G>A/621 + 1G>T . .
4 R553/[Delta]F508 . .
5 G551D/wild-type . .
6 3659delC/[Delta]F508 . .
7 [Delta]I507/wild-type . .
8 711 + 1G>T/621 + 1G>T M* .
9 621 + 1G>T/[Delta]F508 . .
10 G85E/621 + 1G>T . .
11 A455E/[Delta]F508 . .
12 R560T/[Delta]F508 . .
13 N1303K/G1349D . .
14 G542X/G542X m* .
15 W1282X/wild-type . .*
16 2789 + 5G>A/2789 + 5G>A . .
17 3849 + 10C>T/3849 + 10C>T . .
18 1717-1G>T/wild-type . .
19 R1162X/wild-type . .
20 R347P/G551D . .
21 R334W? . .
22 R117H/[Delta]F508 m .
23 2184delA/[Delta]F508 . .
24 1898 + 1G>A/[Delta]F508 . .

 17 18
Sample Allelic Variant 2184delA 3120 + 1G>A

1 Wild-type DNA . .
2 [Delta]F508/ F508 . .
3 3120 + 1G>A/621 + 1G>T . m
4 R553/[Delta]F508 . .
5 G551D/wild-type . .
6 3659delC/[Delta]F508 . .
7 [Delta]I507/wild-type .
8 711 + 1G>T/621 + 1G>T . .
9 621 + 1G>T/[Delta]F508 . .
10 G85E/621 + 1G>T m* .
11 A455E/[Delta]F508 . .
12 R560T/[Delta]F508 . .
13 N1303K/G1349D . .
14 G542X/G542X . .
15 W1282X/wild-type . .
16 2789 + 5G>A/2789 + 5G>A . .
17 3849 + 10C>T/3849 + 10C>T . .
18 1717-1G>T/wild-type . .
19 R1162X/wild-type . .
20 R347P/G551D . .
21 R334W? . .
22 R117H/[Delta]F508 . .
23 2184delA/[Delta]F508 m .
24 1898 + 1G>A/[Delta]F508 . .

 19 20
Sample Allelic Variant 621 + 1G>T [Delta] F508

1 Wild-type DNA . .
2 [Delta]F508/ F508 . M
3 3120 + 1G>A/621 + 1G>T m .
4 R553/[Delta]F508 . m
5 G551D/wild-type . .
6 3659delC/[Delta]F508 . m
7 [Delta]I507/wild-type . .
8 711 + 1G>T/621 + 1G>T m .
9 621 + 1G>T/[Delta]F508 m m
10 G85E/621 + 1G>T m .
11 A455E/[Delta]F508 . m
12 R560T/[Delta]F508 . m
13 N1303K/G1349D . .
14 G542X/G542X . .
15 W1282X/wild-type . .
16 2789 + 5G>A/2789 + 5G>A . .
17 3849 + 10C>T/3849 + 10C>T . .
18 1717-1G>T/wild-type . .
19 R1162X/wild-type . .
20 R347P/G551D . .
21 R334W? . .
22 R117H/[Delta]F508 . m
23 2184delA/[Delta]F508 . m
24 1898 + 1G>A/[Delta]F508 . m

 21 22
Sample Allelic Variant [Delta]507 1898 + 1G>A

1 Wild-type DNA . .
2 [Delta]F508/ F508 . .
3 3120 + 1G>A/621 + 1G>T . .
4 R553/[Delta]F508 . .
5 G551D/wild-type . .
6 3659delC/[Delta]F508 . .
7 [Delta]I507/wild-type m .
8 711 + 1G>T/621 + 1G>T . .
9 621 + 1G>T/[Delta]F508 . .
10 G85E/621 + 1G>T . .
11 A455E/[Delta]F508 . .
12 R560T/[Delta]F508 . .
13 N1303K/G1349D . .
14 G542X/G542X m* .
15 W1282X/wild-type . .
16 2789 + 5G>A/2789 + 5G>A . .
17 3849 + 10C>T/3849 + 10C>T . .
18 1717-1G>T/wild-type . .
19 R1162X/wild-type . .
20 R347P/G551D . .
21 R334W? . .
22 R117H/[Delta]F508 . .
23 2184delA/[Delta]F508 . .
24 1898 + 1G>A/[Delta]F508 . m

 23 24
Sample Allelic Variant 3849 + 10kbC>T 711 + 1G>T

1 Wild-type DNA . .
2 [Delta]F508/ F508 . .
3 3120 + 1G>A/621 + 1G>T . .
4 R553/[Delta]F508 . .
5 G551D/wild-type . .
6 3659delC/[Delta]F508 . .
7 [Delta]I507/wild-type . .
8 711 + 1G>T/621 + 1G>T . m
9 621 + 1G>T/[Delta]F508 . .
10 G85E/621 + 1G>T . .
11 A455E/[Delta]F508 . .
12 R560T/[Delta]F508 . .
13 N1303K/G1349D . .
14 G542X/G542X . .
15 W1282X/wild-type . .
16 2789 + 5G>A/2789 + 5G>A . .
17 3849 + 10C>T/3849 + 10C>T M .
18 1717-1G>T/wild-type . .
19 R1162X/wild-type . .
20 R347P/G551D . .
21 R334W? . .
22 R117H/[Delta]F508 . .
23 2184delA/[Delta]F508 . .
24 1898 + 1G>A/[Delta]F508 . .

(a) Mutation calls for 2 alleles are abbreviated as follows:
dot, wild-type/wild-type; m, wild-type/mutant; M, mutant/mutant; *,
incorrect call; blank, not assayed for the particular mutation in
accordance with the work plan. Mutation panels are defined as
follows: core, 1-24 (using the numbers on the top of table);
Caucasian, M1101K, Y1092X, 2183delAA>G, 3199del6*, 394delTT,
405 + 3A>C; Hispanic, [Delta]F311, D1270N, G330X, I506T, R75X,
S549N, W1089X, Y1092X, 1812-1G>A, 2055del9>A, 2183delAA>G,
3199del6*, 406-1G>A, 935delA; African-American, [Delta]F311,
A559T, G480C, R1066C, R1158X, S1255X, S549N, 1812-1G>A,
2307insA, 405 + 3A C, 444delA, 3791delC. Mutation panels
are based on literature data (14-19). Genotyping results
for Caucasian, Hispanic, and African-American panels
are presented in the online Data Supplement.

The treated multiplex PCR products (10-20 ng of each DNA
fragment) were added to the ASPE reaction mixture, final
volume 40 [micro]L, containing 20 mmol/L Tris-HCl (pH 8.0),
50 mmol/L KCl, 25 nmol/L allele-specific primers,
5 [micro]mol/L biotin-CTP, 0.1% Triton-100, 28 [micro]mol/L
dCTP, 100 [micro]mol/L dNTP ([dCTP.sup.--]), and 3 units Tsp DNA
polymerase. The reactions were incubated at 96 [degrees]C
for 2 min to denature DNA, followed by 30 PCR cycles
(94 [degrees]C for 30 s, 60 [degrees]C for 1 min, and
74 [degrees]C for 2 min) and 72 [degrees]C for 7 min.

Hybridization of MTPs was performed in the 1x prehybridization
buffer [50 mmol/L Tris-HCl, pH 8.0, 150 mmol/L sodium chloride,
0.1% (wt/vol) SDS, 0.5% (wt/vol) Ficoll (type 400), 5 mmol/L
EDTA, pH 8.0, 200 [micro]g/mL sheared, denatured salmon sperm
DNA, 1 [micro]g/[micro]L BSA] at 48 [degrees]C for 10 min.
After removing the prehybridization buffer, the MTPs were
hybridized in 1x hybridization solution (80 [micro]L ASPE
products and 80 [micro]L 2x hybridization buffer) at 48 [degrees]C
for 2 h and rinsed 3 times.

Streptavidin-phycoerythrin conjugate was diluted 1:10 in PBS
(1.06 mmol/L potassium phosphate monobasic, 155.17 mmol/L
sodium chloride, 2.97 mmol/L sodium phosphate dibasic, pH 7.4),
and 10 [micro]L was added to 120 [micro]L 1x washing buffer at a
final concentration of 8 [micro]g/mL. The MTPs bearing the
hybridized DNA were incubated in the above solution for 30 min
at room temperature in the dark, rinsed, and analyzed.
COPYRIGHT 2007 American Association for Clinical Chemistry, Inc.
No portion of this article can be reproduced without the express written permission from the copyright holder.
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Title Annotation:Technical Briefs
Author:Xin, Lin; Flint, James A.; Azaro, Marco; Coradetti, Thomas; Kopacka, Wesley M.; Streck, Deanna L.; Z
Publication:Clinical Chemistry
Date:Jul 1, 2007
Words:3950
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