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Microtransponder-based multiplex assay for genotyping cystic fibrosis.

Cystic fibrosis cystic fibrosis (sĭs`tĭk fībrō`sĭs), inherited disorder of the exocrine glands (see gland), affecting children and young people; median survival is 25 years in females and 30 years in males.  (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 phenylalanine (fĕn'əlăl`ənēn'), organic compound, one of the 22 α-amino acids commonly found in animal proteins. Only the l-stereoisomer appears in mammalian protein.  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 medical genetics
n.
The study of the etiology, pathogenesis, and natural history of diseases and disorders that are at least partially genetic in origin.
 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 DNA: see nucleic acid.
DNA
 or deoxyribonucleic acid

One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes.
 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 (1) (Message Transfer Part) See SS7.

(2) (Media Transfer Protocol) A Microsoft enhancement to the picture transfer protocol (PTP), starting with Windows Media Player 10 in Windows XP.
 (Fig. 1A), a monolithic 500 [micro]m x 500 [micro]m integrated circuit integrated circuit (IC), electronic circuit built on a semiconductor substrate, usually one of single-crystal silicon. The circuit, often called a chip, is packaged in a hermetically sealed case or a nonhermetic plastic capsule, with leads extending from it for  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 (MegaHertZ) One million cycles per second. It is used to measure the transmission speed of electronic devices, including channels, buses and the computer's internal clock. A one-megahertz clock (1 MHz) means some number of bits (16, 32, 64, etc.  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 Oligonucleotide

A deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequence composed of two or more covalently linked nucleotides. Oligonucleotides are classified as deoxyribooligonucleotides or ribooligonucleotides.
 immobilized on the MTP. The current MTP design uses 10 bits to encode (1) To assign a code to represent data, such as a parts code. Contrast with decode.

(2) To convert from one format or signal to another. See codec and D/A converter.

(3) The term is sometimes erroneously used for "encrypt.
 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 hy·drox·y  
adj.
Containing the hydroxyl group.



[From hydroxyl.]


hydroxy  

Containing the hydroxyl group (OH).

Adj. 1.
 and amino groups on the surface of the chip. Such derivatized MTPs are subjected to oligonucleotide synthesis Oligonucleotide synthesis is the non-biological, chemical synthesis of defined short sequences of nucleic acids. It is extremely useful in laboratory procedures covering a wide range of molecular biology applications. .

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 sheared  
adj.
Shaped or finished by shearing, especially cut or trimmed to a uniform length: a sheared fur coat.

Adj. 1.
. 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 Primer extension is a technique whereby the 5' ends of RNA or DNA can be sequenced.

In this technique, we need an oligonucleotide from a transcribed DNA sequence. This oligonucleotide is annealed to the mRNA.
 (ASPE ASPE Assistant Secretary for Planning and Evaluation (US Department of Health and Human Services)
ASPE American Society of Plumbing Engineers
ASPE American Society for Precision Engineering
ASPE Association of Standardized Patient Educators
) (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 allele (əlēl`): see genetics.
allele

Any one of two or more alternative forms of a gene that may occur alternatively at a given site on a chromosome.
 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 DNA polymerase /DNA po·lym·er·ase/ (pah-lim´er-as) any of various enzymes catalyzing the template-directed incorporation of deoxyribonucleotides into a DNA chain, particularly one using a DNA template.  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 biotin: see vitamin; coenzyme.
biotin

Organic compound, part of the vitamin B complex, essential for growth and well-being in animals and some microorganisms.
 moieties, which were subsequently visualized as a result of a binding reaction with phycoerythrin-labeled streptavidin (phycoerythrin phy·co·er·y·thrin  
n.
A red phycobilin occurring especially in the cells of red algae.

Noun 1. phycoerythrin - red pigment in red algae
 is a pigment-carrying, highly fluorescent protein from red algae red algae: see seaweed; Rhodophyta. ). The fluorescence was then read in the flow reader instrument.

Eighteen pairs of PCR PCR polymerase chain reaction.

PCR
abbr.
polymerase chain reaction


Polymerase chain reaction (PCR) 
 primers were designed using an in-house custom software package, SimuPlex (9). SimuPlex accepts a sequence file comprising all the loci loci

[L.] plural of locus.

loci Plural of locus, see there
 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 simulated annealing - A technique which can be applied to any minimisation or learning process based on successive update steps (either random or deterministic) where the update step length is proportional to an arbitrarily set parameter which can play the role of a temperature.  algorithm to search the surrounding solution space for even better multiplex sets. The melting temperature Melting temperature may refer to:
  • Melting temperature, the temperature at which a substance changes from solid to liquid state.
  • DNA melting temperature, the temperature at which a DNA double helix dissociates into single strands.
 ([T.sub.m]) and free-energy calculations are based on the most accurate and up-to-date formulas and thermodynamic ther·mo·dy·nam·ic
adj.
1. Characteristic of or resulting from the conversion of heat into other forms of energy.

2. Of or relating to thermodynamics.
 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 Python, in Greek mythology
Python, in Greek mythology, a huge serpent. In some myths the infant Apollo slew Python at the oracle of Gaea in Delphi; in others Apollo killed the serpent in order to claim the oracle for himself.
: 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 Noun 1. single nucleotide polymorphism - (genetics) genetic variation in a DNA sequence that occurs when a single nucleotide in a genome is altered; SNPs are usually considered to be point mutations that have been evolutionarily successful enough to recur in a  analyzed by ASPE, 2 allele-specific primers (ASP) were synthesized, with each ASP differing in the tag sequence and in the polymorphic polymorphic - polymorphism  nucleotide contained at its 3' terminus Terminus (tûr`mĭnəs), in ancient Rome, both the boundary markers between properties and the name of the god who watched over boundaries. .

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 genomic DNA
n.
The full complement of DNA contained in the genome of a cell or organism.
 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 The University of Medicine and Dentistry of New Jersey is the state-run health sciences institution of New Jersey and comprises eight distinct academic units: the New Jersey Medical School, the New Jersey Dental School, the Graduate School of Biomedical Sciences, the School of . During the course of this project, >100 CF reagent sets were prepared. Each reagent set consisted of a vial vial

a small bottle.
 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 ho·mo·zy·gous
adj.
Having the same alleles at one or more gene loci on homologous chromosome segments.


Homozygous
Identical genes controlling a specified inherited trait.
 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 DNA testing
Analysis of DNA (the genetic component of cells) in order to determine changes in genes that may indicate a specific disorder.

Mentioned in: Acoustic Neuroma, Retinoblastoma, Von Willebrand Disease
, 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 (Digital Object Identifier) A method of applying a persistent name to documents, publications and other resources on the Internet rather than using a URL, which can change over time. : 10.1373/clinchem.2006.081810

References

(1.) Palomaki GE, Fitzsimmons SC, Haddow JE. Clinical sensitivity of prenatal prenatal /pre·na·tal/ (-na´tal) preceding birth.

pre·na·tal
adj.
Preceding birth. Also called antenatal.



prenatal

preceding birth.
 screening for cystic fibrosis via CFTR carrier testing in a United States United States, officially United States of America, republic (2005 est. pop. 295,734,000), 3,539,227 sq mi (9,166,598 sq km), North America. The United States is the world's third largest country in population and the fourth largest country in area.  panethnic population. Genet genet: see civet.  Med 2004;6:405-14.

(2.) American College of Obstetrics and American College of Medical Genetics. Preconception pre·con·cep·tion  
n.
An opinion or conception formed in advance of adequate knowledge or experience, especially a prejudice or bias.

Noun 1.
 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 (Radio Frequency IDentification) A data collection technology that uses electronic tags for storing data. The tag, also known as an "electronic label," "transponder" or "code plate," is made up of an RFID chip attached to an antenna.  electronic chips, as platforms for cell growth in cytotoxicity cytotoxicity /cy·to·tox·ic·i·ty/ (si?to-tok-sis´i-te) the degree to which an agent possesses a specific destructive action on certain cells or the possession of such action.  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 The human genome is the genome of Homo sapiens, which is composed of 24 distinct pairs of chromosomes (22 autosomal + X + Y) with a total of approximately 3 billion DNA base pairs containing an estimated 20,000–25,000 genes. . Science 1988;280:1077-82.

(8.) Gerry NP, Witowski NE, Day J, Hammer RP, Barany G, Barany F. Universal DNA microarray DNA microarray

A small solid support, usually a membrane or glass slide, on which sequences of DNA are fixed in an orderly arrangement. DNA microarrays are used for rapid surveys of the expression of many genes simultaneously, as the sequences contained on a
 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 "Not invented here." See digispeak.

NIH - The United States National Institutes of Health.
 Small Business Innovation Research Conference, July 2005, Bethesda, MD.

(10.) SantaLucia JJ, Hicks Hicks   , Edward 1780-1849.

American painter of primitive works, notably The Peaceable Kingdom, of which nearly 100 versions exist.
 D. The thermodynamics thermodynamics, branch of science concerned with the nature of heat and its conversion to mechanical, electric, and chemical energy. Historically, it grew out of efforts to construct more efficient heat engines—devices for extracting useful work from expanding  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 See Control Data, century date change and Back Orifice.

CDC - Control Data Corporation
 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.

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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 molecular genetics
n.
The branch of genetics that deals with hereditary transmission and variation on the molecular level.
, University of Medicine and Dentistry, New Jersey-New Jersey Medical School, Newark, NJ;

* address correspondence to this author at: PharmaSeq, Inc., 11 Deer Park Deer Park.

1 Uninc. village (1990 pop. 28,840), Babylon town, Suffolk co., SE N.Y., a primarily residential suburb on Long Island.

2 City (1990 pop. 27,652), Harris co., SE Tex.
 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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