Annals of Clinical & Laboratory Science 33:243-250 (2003)
© 2003 Association of Clinical Scientists
Single Tube Multiplex PCR Detection of 27 Cystic Fibrosis Mutations and 4 Polymorphisms using Neonatal Blood Samples Collected on Guthrie Cards
Gregory S Makowski,
Francesca L Nadeau and
Sidney M Hopfer
Department of Laboratory Medicine, University of Connecticut Health Center, Farmington, Connecticut
Address correspondence to: Gregory S. Makowski, Ph.D., Department of Laboratory Medicine, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-2235, USA; tel: 860 679 2596; fax: 860 679 2154; e-mail: makowski{at}nso1.uchc.edu.
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Abstract
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In response to recommendations for cystic fibrosis (CF) carrier screening of the American College of Medical Genetics/American College of Obstetrics and Gynecology (ACMG/ACOG), we evaluated a commercial kit for mutation panel testing (Roche CF Gold Linear Array Panel). This kit tests for 25 CF mutations and 4 polymorphisms; it comprises an analyte specific reagent for single tube multiplex polymerase chain reaction (PCR) amplification and subsequent allele specific oligonucleotide (ASO) hybridization. Neonatal whole blood samples collected on Guthrie card filter paper served as the DNA source. Following a wash step to remove whole blood PCR inhibitors, multiplex PCR amplification could be performed either on DNA that was heat extracted from Guthrie cards or directly on the filter paper matrix itself. In 13 CF patient samples, the CF mutation results obtained with this kit agreed completely with those obtained from a reference laboratory that performs an 87 CF mutation panel. The kit was reliable, despite the small sample size (3 mm diameter punch of the Guthrie card), the presence of PCR inhibitors in whole blood, and protracted storage of blood samples (up to 9 yr at room temperature). The kit was convenient, cost competitive, and practical for use in a small CF screening laboratory.
(received 25 March 2003; accepted 30 March 2003)
Keywords: multiplex PCR, cystic fibrosis, mutation analysis
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Introduction
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Cystic fibrosis (CF) is the most common autosomal recessive genetic disorder; the reported incidence is 1:3,200 in Caucasian infants [1]. Since the identification in 1989 of the cystic fibrosis transmembrane conductance regulator (CFTR) gene [2,3], >1000 CF mutations and polymorphisms have been documented [4]. The American College of Medical Genetics (ACMG) has recommended that CF genetic screening tests consist of an abbreviated, but core panel of 25 mutations and 4 reflex test polymorphisms, based on pan-ethnic CF mutation frequency [5]. These recommendations have also been adopted by the American College of Obstetrics and Gynecology [6].
The Cystic Fibrosis Newborn Screening Laboratory of the University of Connecticut Health Center performs immunoreactive trypsinogen (IRT) testing of approximately one-half of the neonates born in the State of Connecticut [7]. Approximately, 20,000 IRT tests are performed annually using whole blood samples collected on Guthrie card filter paper at the time of hospital discharge and at the two-week follow-up appointment with the pediatrician, if necessary (see below). Discharge samples that are highly positive for IRT (>150 ng/ml) are immediately subjected to polymerase chain reaction (PCR) amplification for the deltaF508 mutation using a method developed in our laboratory [8–10]. In subjects with clinical symptoms, discharge blood samples that are positive for IRT (>90 ng/ml) are tested by PCR amplification for the deltaF508 mutation; these subjects also undergo CF sweat tests. IRT analysis is performed on the two-week sample if the discharge sample gives a positive IRT result (>90 ng/ml) in subjects without a family history or clinical symptoms of CF. Two-week samples that give IRT results >70 ng/ml are subjected to PCR amplification for the deltaF508 mutation; the subjects also undergo CF sweat testing. If the genetic testing in our laboratory is negative, the blood samples are sent to a reference laboratory for analysis using an 87 CF mutation panel.
In response to the ACMG/ACOG recommendations [5,6], we evaluated a commercial kit for detection of 27 CF mutations and 4 polymorphisms using single tube multiplex PCR amplification and ASO hybridization (Roche CF Gold Linear Array Panel).
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Materials and Methods
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Materials.
Whole blood samples were collected from neonates by heelstick on Guthrie card filter paper (#903, Schleicher & Schuell, Inc., Keene, NH). The multiplex polymerase chain reaction (PCR) and allele specific oligonucleotide (ASO) hybridization kit for 27 CF mutations and 4 polymorphisms (Table 1
) used an analyte specific reagent (Linear Array CF Gold 1.0, Roche Molecular Systems, Inc., Indianapolis, IN). DNA molecular weight 100 bp ladders (1 mg/ml) were from Invitrogen Corp. (Carlsbad, CA). Electrophoretic grade acrylamide, N,N’-methylene-bis-acrylamide, and all other reagents were from Sigma-Aldrich, Inc. (St. Louis, MO). Multiplex PCR kit components and other reagents were stored as recommended by the manufacturers. This study protocol was approved by the Institutional Review Board, Office of Research Compliance, University of Connecticut Health Center (IRB #03-099).
DNA extraction.
Whole blood samples were collected from neonates on Guthrie card filter paper, air dried, and stored at room temperature as previously described [11]. The storage periods for bloodspot samples ranged from 1 to 109 mo. To remove endogenous whole blood PCR inhibitors [12–14], bloodspots were prepared by two methods. In Method 1 (modified in-house method) [8–10], a single bloodspot (3 mm diameter) was washed twice in 0.5 ml ultrapure water (20 min each). In Method 2 (courtesy of G. Hoffman, Newborn Screening Laboratory, Madison, WI), 2 bloodspots (3 mm diameter) were rapidly washed twice in 75 µl of phosphate buffered saline containing 8 mM Na2HPO4, 2 mM KH2PO4, 138 mM NaCl, and 3 mM KCl (3 min each). In both methods the washes were pooled and saved for protein analysis. To each bloodspot was then added 75 µl of DNA extraction buffer containing 10 mM Tris-Cl, pH 8.3, and 50 mM KCl, and the tubes were heated at 96°C for 30 min. After heating, an aliquot of the DNA extract (25 µl) was used for multiplex PCR amplification. To evaluate the presence of residual DNA on the washed/heated filter matrix, a single washed bloodspot (obtained using method 1) was placed in a PCR tube (with 25 µl ultrapure water to adjust for volume) and multiplex PCR amplification was performed directly.
Protein analysis.
In bloodspot washes obtained from DNA extraction by methods 1 and 2 (see above) protein concentration was assayed by a pyrogallol red dye binding technique [15] using an automated analyzer (Synchron LX20, Beckman-Coutler, Inc, Brea, CA).
Multiplex PCR amplification.
Multiplex PCR amplification was performed using 25 µl of extracted DNA (see methods 1 and 2 above) or a washed 3 mm bloodspot punch (with 25 µl ultrapure water added). To these samples was added 50 µl of PCR master mix and 25 µl of 32 mM MgCl2 to achieve a final volume of 100 µl. The PCR master mix was prepared just prior to use and contained the following final concentrations: 10 mM Tris-Cl, 50 mM KCl, 0.12 mM 5'-biotinylated primer mix, 0.3 mM dATP, 0.3 mM dCTP, 0.3 mM dGTP, 0.6 mM dUTP, 3 U uracil N-glycosylase, 15 U FastStart Taq DNA polymerase, and 8 mM MgCl2. The recipe for the appropriate volume of PCR master mix is shown (Table 2).
Multiplex PCR amplification was performed using a GeneAmp PCR System 9600 thermal cycler (Applied Biosystems, Inc., Foster City, CA). Samples were amplified as follows: hold for 10 min at 42°C (to activate uracil N-glycosylase); hold for 6 min at 93°C (to inactivate uracil N-glycosylase); and then cycled (32 cycles) as follows: 30 sec at 93°C (denaturation); 30 sec at 60°C (annealing); 1 min at 72°C (elongation); and then hold for up to 2 hr at 15°C. Following amplification, samples could be used immediately for ASO hybridization or stored at -10°C.
ASO hybridization.
Hybridization strips (4 mm x 13 cm) contained a total of 55 different allele specific oligonucleotide probes to normal and CF mutation sequences (Linear Array Panels). Strips were placed in strip-type incubation wells and 6 ml of prewarmed (37°C) hybridization/wash buffer (H/W buffer) containing 30 mM NaH2PO4, pH 7.4, 0.54 M NaCl, 3 mM Na2EDTA, and 0.5% (w/v) sodium dodecylsulfate (SDS) was added. An aliquot of each multiplex PCR sample (60 µl) was quickly mixed with 60 µl of denaturation solution containing 0.4 M NaOH and 20 mM Na2EDTA.
The denatured sample (100 µl) was immediately added to each hybridization strip in H/W buffer. The strip tray was covered and incubated with gentle mixing for 25 min at 50°C in a water bath (Model 25, Precision Scientific Instruments, Inc., Chicago, IL). Following ASO hybridization, the strips were aspirated, washed with 5 ml warm (37°C) H/W buffer, and re-aspirated. Conjugate solution containing 5 ml of warm (37°C) H/W buffer and 20 µl of strepavidin-conjugated horseradish peroxidase (prepared just prior to use) was added to each strip and incubated with gentle mixing for 25 min at 50°C.
The strips were then aspirated, washed with 5 ml of warm (37°C) H/W buffer, aspirated, washed with 5 ml of room temperature citrate buffer containing 100 mM sodium citrate, pH 5.0, and again aspirated. Substrate solution (room temperature) containing 5 ml of citrate buffer, 5 µl of 3% H2O2, and 250 µl of 3,3',5,5'-tetramethyl-benzidine (2 mg/ml absolute ethanol, prepared 30 min prior to use) was then added to each strip.
Color development was visually monitored and allowed to proceed for 10–20 min at room temperature, at which time the substrate solution was aspirated. The strips were then washed twice with 3 ml water (about 5 min each), carefully removed from the incubation wells, and gently blotted dry with absorbent paper. Hybridization strips were then read using the strip guide aligned with the CF Gold 1.0 Mutation Reference Guide.
For recording purposes, strips were affixed with tape to cardstock paper and photocopied or digitally scanned (ScanJet 6100C, Hewlett-Packard Instruments, Inc). Strips affixed to cardstock paper could be stored in polypropylene sheet protectors at room temperature without appreciable loss of band intensity.
Cystic fibrosis mutations detected by this protocol were previously characterized by our assay for the deltaF508 mutation [8–10] or by an extensive mutation analysis (CF87 Mutation Panel) by referral to Genzyme Genetics, Inc. (Framingham, MA).
Quality control.
Three quality control materials (negative, normal, and abnormal) were routinely amplified. The negative control consisted of addition of 25 µl of water to the amplification mix. No visible amplification products should be detected on the hybridization strip. The normal control consisted of a bloodspot collected from a normal (ie, non cystic fibrosis) individual. Only normal alleles should be detected on the hybridization strip.
The abnormal control consisted of a bloodspot collected from a previously characterized subject with homozygous deltaF508 cystic fibrosis. The deltaF508 mutation should be the only mutation detected on the hybridization strip; all other alleles should be normal.
Electrophoresis.
Tris-borate-EDTA/polyacrylamide gel electrophoresis (TBE-PAGE) was performed as previously described [8]. Briefly, the multiplex PCR samples (10 µl) were mixed with 2 µl of 6X sample buffer containing bromophenol blue and Ficoll-type 400. Samples (5 µl) were electrophoresed on 4% mini-polyacrylamide gels (0.75 mm thickness, 6 cm height) (Bio-Rad Laboratories, Inc., Hercules, CA) in 1X TBE at 75 V (constant voltage) for 1 hr at room temperature [8].
Gels were stained with ethidium bromide (0.5 µg/ml) in 100 ml of 1X TBE for 10 min and photographed with background UV illumination (Spectronics Corp., Westbury, NY). Electrophoretic migration of multiplex amplicons was compared to DNA molecular weight 100 bp ladders (1 µg/lane).
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Results
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To eliminate whole blood PCR inhibitors [9,12–14], Guthrie card bloodspots were processed by an extended wash (Method 1) or a rapid wash procedure (Method 2) prior to DNA extraction. Template DNA was then subjected to multiplex PCR amplification for 27 cystic fibrosis mutations and 4 polymorphisms using the CF Gold 1.0 Primer Mix and amplicons were hybridized to allele specific oligonucleotides immobilized in Linear Array Panels (Fig. 1).
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Fig. 1. Multiplex PCR hybridization results. DNA was extracted from Guthrie card bloodspots by two methods and multiplex PCR amplification performed with 5'-biotinylated primers. Amplicons were denatured, hybridized to allele specific oligonucleotides immobilized on plastic strips (Linear Array Panels), probed with strepavidin-conjugated horseradish peroxidase, and visualized with TMB substrate. DNA was prepared using our laboratory’s routine procedure (Method 1) or a rapid wash procedure (Method 2). Amplification was also performed directly on a washed filter paper disk from patient #2 (C). The patient sample number is indicated above each hybridization strip; subject #5 is normal (non-CF). Hybridization strips were aligned with the Roche CF Mutation Guide (on right). Mutation results for patients #1 to #4 (and other patients) are listed in Table 3 .
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The 15 primer sets were all successfully amplified and hybridization results for both normal and mutant alleles completely agreed using both wash methods. Some hybridization bands were, however, slightly less intense with Method 2 (rapid wash procedure). This finding probably resulted from retention of PCR inhibitors, since more contaminants (eg, protein) were released with the extended wash procedure (Fig. 2). Direct use of the washed filter paper indicated that residual filter-bound DNA was sufficient for multiplex PCR amplification (lane C, Fig. 1).
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Fig. 2. Protein release from Guthrie card bloodspots. Filter punches (3 mm diameter) were washed twice (20 min each) with 500 µl of water (extended wash, Method 1) or twice (3 min each) with 75 µl of PBS (rapid wash, Method 2) prior to extraction of DNA for multiplex PCR amplification. The washes were pooled and protein determinations performed. Protein release is an index of the removal of whole blood contaminants that interfere with PCR amplification [9,12–14]. The storage period (mo) of the bloodspot is indicated above each data set. These data were obtained from samples #1 to #5 (see Fig. 1).
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The presence of multiplexed PCR amplicons was confirmed by electrophoresis on polyacrylamide gels (TBE-PAGE) (Fig. 3). Ethidium bromide staining demonstrated significant quantities of about 12 amplified products for each multiplex PCR reaction. The presence of these PCR products at an apparent electrophoretic mobility of 100–600 bp was consistent with expected amplicon size (Table 1). No substantial quantitative or qualitative differences were observed between the two wash methods or when the filter matrix was amplified directly (Fig. 3). Although the electrophoretic analysis was unable to resolve the 15 individual primer-specific amplicons, it provided a rapid means (about 1 hr) to confirm the presence of amplified product and it could be performed on a small volume of sample (5 µl) prior to the ASO hybridization.
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Fig. 3. TBE-PAGE of multiplex PCR amplicons. Multiplex PCR amplified samples were electrophoresed on 4% TBE-PAGE. DNA was prepared by our routine procedure (Method 1) or by a rapid wash procedure (Method 2). Amplification was also performed directly on a washed filter paper disk from patient #2 (C). The patient sample number is indicated above each hybridization strip. Sample #5 is normal (non-CF). The DNA 100 bp molecular weight (Mw) ladder is shown at left. Samples were diluted 1:6 with sample buffer and 5 µl loaded per lane; the gel was stained with ethidium bromide.
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Results of multiplex PCR amplification with ASO hybridization were obtained from a total of 13 Guthrie card samples from CF patients (Table 3). The periods of sample storage ranged from 1 to 109 mo. All of the CF mutation and polymorphism results were confirmed by assays in the reference laboratory. As can be seen, most CF patients were compound heterozygotes with a least one copy of the deltaF508 mutation. Two CF patients with the R117H mutation also possessed 5T polymorphism (patients #5 and #13). The mutation status of two confirmed CF patients (sweat chloride >60 mEq/L) remained unknown: one of these patients (#8) had a single copy of deltaF508; in the other patient (#7) no mutation was identified by multiplex PCR amplification using the Linear Array Panels or by the 87 CF mutation panel assay in the reference laboratory.
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Discussion
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Although cystic fibrosis (CF) is the most common autosomal recessive genetic disease in North America [16], genetic screening for CF is complicated by (a) the large number of mutations that have been identified, (b) the variations of mutation prevalence in our heterogeneous population [17], (c) variability in phenotypic expression, and (d) inconsistent clinical presentation of the disease [5].
To address these concerns, the American College of Medical Genetics (ACMG) and the American College of Obstetrics and Gynecology (ACOG) proposed a core mutation panel of 25 CF mutations and 4 reflex tests for polymorphisms for CF carrier screening [5,6]. The ACMG recognized that this recommendation for CF mutation testing could prove difficult for small CF screening sites that do not routinely test for large panels of mutations [5].
In response to the ACMG recommendations, our laboratory evaluated a commercial kit for testing the ACMG/ACOG panel of CF mutations (Roche CF Gold Linear Array Panel). This kit is distributed as an analyte specific reagent for single tube multiplex PCR amplification and subsequent ASO hybridization. In our evaluation, the Roche CF Gold Linear Array Panel was easy to perform and reliable. The CF mutation results were rapidly obtained since the multiplex PCR amplification and ASO hybridization could be performed on 5 to 20 samples in a single day, including sample/reagent preparation (1 hr), PCR amplification (2.5 hr), TBE-PAGE analysis (if desired, 1 hr), and ASO hybridization (2 hr).
Because of the presence of whole blood PCR inhibitors [9,12–14], we recommend the extended wash procedure for Guthrie card bloodspots using water or a relatively low concentration of buffer such as PBS. We found the kit method to be robust since sufficient template DNA for multiplex PCR amplification can be performed with 25 µl of sample obtained from a single bloodspot (3 mm diameter) and heat extracted into 75 µl of buffer (96°C, 30 min). Alternatively, PCR amplification can be performed directly on a single bloodspot, from filter matrix immobilized DNA as shown here and previously by our laboratory [9,11].
Because DNA in dried bloodspots appears to be highly stable, others have suggested that stored Guthrie cards provide a unique and convenient repository of genetic information [18,19]. In this study, multiplex PCR amplification was successfully performed on bloodspots that had been stored at room temperature for as long as 109 mo (approximately 9 yr). Following ASO hybridization, the linear array panels can be stored indefinitely in polypropylene folders or digitally scanned for documentation purposes. The CF mutation results that were obtained using this kit were consistent with the results obtained by a large reference laboratory for genetic screening.
In conclusion, we found the Roche CF Gold 1.0 Linear Array Panel kit to be well adapted for use in a small CF screening laboratory. Although some additional equipment was needed (eg, thermally controlled water baths), such equipment is generally inexpensive and readily available in clinical laboratories that perform molecular testing. The procedure is labor intensive, but we found that it was cost effective, compared to cost of sending the samples for testing in a reference laboratory. Personnel with previous molecular diagnostic experience (PCR amplification) generally required minimal training; the reagent preparation was straightforward, and the CF mutations were readily identified by alignment with the reference guide. Thus, the Roche CF Gold 1.0 Linear Array Panel enables small laboratories, such as ours, as well as larger facilities [16], to implement the recommended ACMG/ACOG guidelines for testing a 25 CF mutation panel with reflex polymorphisms.
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Acknowledgements
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The authors certify that there were no conflicts of interest in regard to this study.
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References
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- Hamoush A, Fitzsimmons SC, Macek M, Knowles MR, Rosenstein BJ, Cutting GR. Comparison of the clinical manifestations of cystic fibrosis in black and white patients. J Pediatr 1998;132:255–259.[Medline]
- Rommens JM, Iannuzzi MC, Kerem B-S, Drumm ML, Melmer G, Dean M, Rozmahel R, Cole JL, Kennedy D, Hidaka N. Identification of the cystic fibrosis gene: chromosome walking and jumping. Science 1989;245: 1059–1065.[Abstract/Free Full Text]
- Kerem B-S, Rommens JM, Buchanan JA, Markiewicz D, Cox TK, Chakravarti A, Buchwald M, Tsui L-C. Identification of the cystic fibrosis gene: genetic analysis. Science 1989;245:1073–1080.[Abstract/Free Full Text]
- Strom CM, Huang D, Chen C, Buller A, Peng M, Quan F, Redman J, Sun W. Extensive sequencing of the cystic fibrosis transmembrane regulator gene: assay validation and unexpected benefits of developing a comprehensive test. Genet Med 2003;5:9–14.[Medline]
- Grody WW, Cutting GR, Klinger KW, Richards CS, Watson MS, Desnick RJ. Laboratory standards and guidelines for population-based cystic fibrosis carrier screening. Genet Med 2001;3:149–154.[Medline]
- American College of Obstetrics and Gynecology and American College of Medical Genetics. Preconception and Prenatal Carrier Screening for Cystic Fibrosis; Clinical and Laboratory Guidelines. American College of Obstetrics and Gynecology, Washington, DC, 2001, pp 1–18.
- Makowski GS, Hopfer SM. Cystic fibrosis: molecular approaches to diagnosis. Ann Clin Lab Sci 1998;28:380–385.[Abstract]
- Makowski GS, Aslanzadeh J, Hopfer SM. Mini-gel PAGE for enhanced resolution of polymerase chain reaction detection of deltaF508 deletion in cystic fibrosis. Clin Chem 1993;39:2204–2205.[Free Full Text]
- Makowski GS, Davis EL, Hopfer SM. Enhanced direct amplification of Guthrie card DNA following selective elution of PCR inhibitors. Nucl Acids Res 1995;23:3788–3789.[Free Full Text]
- Makowski GS, Aslanzadeh J, Hopfer SM. In situ PCR amplification of Guthrie card DNA to detect cystic fibrosis mutations. Clin Chem 1995;41:447–449.
- Makowski GS, Davis EL, Hopfer SM. The effect of storage on Guthrie cards: implications for deoxyribonucleic amplification. Ann Clin Lab Sci 1996;26:458–469.[Abstract]
- Walsh PS, Metzger DA, Higuchi R. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. BioTechniques 1991;10:506–513.[Medline]
- Raskin S, Phillips JA III, Dawson E, Kaplan G, McClure M, Vnencak-Jones C. Cystic fibrosis genotyping by direct PCR analysis of Guthrie card blood spots. PCR Meth Appl 1992;2:154–156.[Medline]
- Jinks DC, Minter M, Tarver DA, Vanderford M, Hejtmancik JF, McCabe ERB. Molecular genetic diagnosis of sickle cell disease using dried blood specimens on blotters used for newborn screening. Hum Genet 1989; 81:363–366.[Medline]
- Fujita Y, Mori I, Kitano S. Color reaction between pyrogallol red-molybdenum (VI) complex and protein. Bunsaki Kagaku 1983;32:E379–386.
- Strom CM, Huang D, Buller A, Redman J, Crossley B, Anderson B, Entwistle T, Sun W. Cystic fibrosis screening using the College panel: platform comparison and lessons learned from the first 20,000 samples. Genet Med 2002; 4:289–296.[Medline]
- Heim RA, Sugarman EA, Allitto BA. Improved detection of cystic fibrosis mutations in the heterogeneous U.S. population using an expanded, pan-ethnic panel. Genet Med 2001;3:168–176.[Medline]
- Guthrie R. Organization of a regional newborn screening laboratory. In: Neonatal Screening for Inborn Errors of Metabolism, (Bickel H, Guthrie R, Hammersen G, Eds), Springer-Verlag, New York, 1980; pp 259–270.
- McEwen JE, Reilly PR. Stored Guthrie cards as DNA "banks". Am J Hum Genet 1994;55:196–200.[Medline]
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Abstract
Introduction
