Polymerase chain reaction amplification from dried blood spots on Guthrie cards

Schwartz, E.I.; Khalchitsky, Sergei E; Eisensmith, Randy C.; Woo, Savio L. C.

doi:10.1016/0140-6736(90)93446-v

Cited by 37 publications

(25 citation statements)

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“…The microextraction procedure, previously described by Jinks et al (10) and used in the study of the 75 unknown specimens in this report, is extremely personnel intensive and, therefore, would be a costly component of any high-volume DNA diagnostic program based on dried blood spots (33). The procedure described by Schwartz et al (26) for direct amplification from a small piece cut from a Guthrie card, without microextraction, would decrease dramatically the personnel investment required for specimen processing. This method was based on the demonstration that DNA could be amplified after boiling the dried blood specimens (10) or directly by the thermostable DNA polymerase from Therrnus therrnophilus (26,34) and was adapted for use with Tag polymerase.…”

Section: Discussionmentioning

confidence: 99%

“…The procedure described by Schwartz et al (26) for direct amplification from a small piece cut from a Guthrie card, without microextraction, would decrease dramatically the personnel investment required for specimen processing. This method was based on the demonstration that DNA could be amplified after boiling the dried blood specimens (10) or directly by the thermostable DNA polymerase from Therrnus therrnophilus (26,34) and was adapted for use with Tag polymerase. This adaptation permits the reduction of sample size from a %-inch diameter semicircle, representing the dried equivalent of approximately 25 pL whole blood, to a 4 X 4 mm square containing approximately 8 pL dried whole blood equivalent.…”

Section: Discussionmentioning

confidence: 99%

“…These attributes have been used not only for genotyping in the hemoglobinopathies (10-14), but also for PKU (6,7,26), CF (8, 9, 18, 19), Duchenne muscular dystrophy (1 5-17), medium-chain acyl CoA dehydrogenase deficiency (20), and hereditary fructose intolerance (2 1). Experience is showing that these specimens can be used for the same molecular genetic analyses that have been applied to liquid blood specimens.…”

Section: Discussionmentioning

confidence: 99%

“…Although the method described above for microextraction and amplification was used pA Probe pS Probe for all of the original 75 blinded specimeni obtained from the Texas Newborn Screening Laboratory in Austin, subsequently we adapted a method from Schwartz et al (26) and previous work on boiling of specimens from our group (10) that avoided the need for manual microextraction. Aliquots measuring approximately 4 x 4 mm (representing the dried equivalent of approximately 8 pL whole blood) were cut from each dried blood specimen and placed in 1.5-mL microfuge tubes to which the complete amplification cocktail including primers, and in some cases Tag polymerase, were added as described above.…”

Section: (5'-ctcaaagaacctctgggtcc-3')mentioning

confidence: 99%

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Genotypic Confirmation from the Original Dried Blood Specimens in a Neonatal Hemoglobinopathy Screening Program

et al. 1992

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ABSTRACT. Dried blood spots are used for newborn screening because of ease of sample collection, handling, and shipment. DNA is stable and accessible in the filter paper matrix. Genotypic confirmation using initial specimens is demonstrated for a regional screening program. Seventy-five blinded samples underwent DNA analysis after Hb electrophoresis. DNA was microextracted from a %-inch semicircle (25 pL whole blood equivalent), amplified, and analyzed by four different methods. Direct amplification without microextraction and automated sequencing from microextracted DNA also was performed. All four analyses agreed for the A and S alleles in 70 of 75 specimens. Three disagreements were clarified by the other semicircle from the original sample: two were due to polymerase chain reaction contamination and one to contamination of one of four analytical tests. Two would have required analysis of a second specimen, one because of polymerase chain reaction failure and the second because the patient had SIB-thalassemia. Direct amplification without microextraction was successful in an additional 77 of 78 specimens for analysis of the A, S, C, and E alleles. Automated direct sequencing from microextracted DNA was demonstrated for the A, S, and C alleles. Analysis of microextracted DNA from dried blood specimens for A and S alleles reduced the need for and costs of obtaining a second specimen for confirmation by 97%. Direct amplification without microextraction for analysis of A, S, and C alleles permits additional reduction in personnel time and costs. We have demonstrated that microextracted DNA is amenable to automated sequencing after asymmetric polymerase chain reaction. Direct genotypic confirmation can facilitate diagnosis and initiation of medical intervention. (Pediatr Res 31: 217-221,1992) Abbreviations PCR, polymerase chain reaction PKU, phenylketonuria CF, cystic fibrosis ASO, allele specific oligonucleotide HRP, horseradish peroxidaseThe collection of newborn screening specimens on filter paper blotters represented a major innovation that facilitated the establishment and regionalization of screening programs (1, 2). These screening programs were originally developed for PKU, but are incorporating an expanding group of disorders, including the hemoglobinopathies (3). With rare exceptions, these expanded test batteries have continued to use dried blood specimens in a filter paper matrix because of the ease of sample collection, handling, and shipment and the stability of the analytes. Typically, the analytes evaluated by neonatal screening laboratories have included metabolites such as phenylalanine for PKU, or galactose and galactose-1-phosphate for galactosemia; hormones such as thyroid hormone and thyroid stimulating hormone (TSH) for congenital hypothyroidism, or 17-hydroxyprogesterone for congenital adrenal hyperplasia; and proteins such as galactose-1-phosphate uridyl transferase for galactosemia, immunoreactive trypsinogen for CF, or Hb for the hemoglobinopathies (1, 3, 4).More recently, it has beco...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: (5'-ctcaaagaacctctgggtcc-3')mentioning

confidence: 99%

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Genotypic Confirmation from the Original Dried Blood Specimens in a Neonatal Hemoglobinopathy Screening Program

et al. 1992

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“…All newborn blood samples were obtained with approval from the State of California Health and Welfare Agency Committee for the Protection of Human Subjects. Genomic DNA was extracted from dried newborn screening blood spots on filter paper using Puregene DNA Purification kit (Gentra, Minneapolis, MN) according to the manufacturer's instructions and was amplified using PCR (Schwartz et al, 1990). …”

Section: Study Subjectsmentioning

confidence: 99%

Screening for novel PAX3 polymorphisms and risks of spina bifida

Lü

Zhu

Wen

et al. 2006

Birth Defects Research

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BACKGROUND: PAX3 plays an important role in mammalian embryonic development. Known mutations in PAX3 are etiologically associated with Waardenburg syndrome and syndromic neural tube defects (NTDs). Mutations in the murine homologue, pax3, are responsible for the phenotype of splotch mice, in which nullizygotes are 100% penetrant for NTDs. METHODS: The study sample included 74 infants with spina bifida (cases) and 87 nonmalformed infant controls. The conserved paired-box domain as well as the upstream genomic region of PAX3 were subjected to resequencing and those identified SNPs were evaluated as haplotypes. The associations of haplotypes for selected gene regions and the risks of spina bifida were further studied. RESULTS: Nineteen SNPs were observed; 15 observed in controls had been submitted to the National Center for Biotechnology Information (NCBI) database with allele frequencies. The PAX3 gene variant T-1186C (rs16863657) and its related haplotype, TCTCCGCCC of nine SNPs, were found to be associated with an increased risk of spina bifida, with an OR of 3.5 (95% CI: 1.2-10.0) among Hispanic Whites. CONCLUSIONS: Our analyses indicated that PAX3 SNPs were not strong risk factors for human spina bifida. However, additional follow-up of the PAX3 gene variant T-1186C (rs16863657) and its related haplotype, TCTCCGCCC, may be important in other populations.

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