1WISCONSIN STATE LABORATORY OF HYGIENEWISCONSIN STATE LABORATORY OF HYGIENE
Molecular LDT in Newborn Screening Laboratories
APHL/CDC Newborn Screening Molecular Workshop Atlanta, GA
June 28-30, 2011
Mei Baker, M.D., FACMG
Assistant Professor, Department of PediatricsUniversity of Wisconsin School of Medicine and Public Health
Science Advisor, Newborn Screening LaboratoryWisconsin State Laboratory of Hygiene
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Outline
• Background information on the disorder
• Knowledge of the gene and disease-causing mutations
• Assay development and validation
– PCR in general– TETRA-primer ARMS-PCR----GALT mutation analysis– Rea-time qPCR----screening for SCID by quantitating
TRECs
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Information Resources_NCBI
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NCBI_OMIM
Information includes:Clinical Features DiagnosisClinical Management PathogenesisMolecular GeneticsGenotype/Phenotype CorrelationsPopulation Genetics
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NCBI_PubMed
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NCBI_Gene
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NCBI_Gene
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Information Resources_GeneTests
Information includes:SummaryDiagnosisClinical DescriptionDifferential DiagnosisManagementGenetic CounselingMolecular GeneticsResources
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HGMD_GALT
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GALT Mutation Map
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General Guidelines for PCR_1
• Preventing contamination– Cross-contamination– Carryover contamination
• Important reaction components– DNA polymerase– Templates– Primers– Deoxynucleoside triphosphate (dNTPs)– Magnesium ions
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General Guidelines for PCR_2
• Cycling parameters– Denaturing– Annealing– Elongation
• PCR products detection– Agarose gel electrophoresis
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Utilities of Molecular Testing in NBS
• Molecular assay as the second tier testing
biochemical metabolites or enzyme activities can be influenced by feeding history, sampling time and environmental factors.
Timely gene mutation information can be helpful in disease management.
• Molecular assay as the primary screening testing
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Utilities of Molecular Testing in NBS: Examples
• GALT gene mutations are responsible for classic galactosemia, and the common mutations are Q188R, S135L, K285N, L195R, Fl71S and Y209C.
• N314D Duarte variant.
• BCKDHA Y438N is only MSUD mutation in the Old Order Mennonite population, which is increasing in the state of Wisconsin. In this population, classic MSUD has a frequency as high as 1 in 176 births
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15Copyright restrictions may apply.
Ye, S. et al. Nucl. Acids Res. 2001 29:e88; doi:10.1093/nar/29.17.e88
Scheme of tetra-primer ARMS-PCR
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Primer Design http://cedar.genetics.soton.ac.uk/public_html/primer1.htm
Source sequence (up to 1,000 bases)
Position of SNP from start of sequence
Allele 1
Allele 2
Optimum (inner) product size
Maximum (inner) product size
Minimum (inner) product size
Maximum relative size difference of two inner products
Minimum relative size difference of two inner products
Shu Ye, Nucleic Acid Research, 2001, Vol. 29, No. 17, E88-8
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Tetra-primer ARMS-PCR Reaction
• Reaction Mix: (25 µl)
1X PCR buffer Forward inner primer 10 pmol Reverse inner primer 10 pmol Forward outer primer 1 pmol Reverse outer primer 1 pmol DNTPs 200 µM MgCl2 2.5 mM Taq DNA polymerase 2.5 U Genomic DNA 4 µl
• Thermal Cycler Condition
1. 95ºC for 5 minutes 2. 95ºC for 30 second 3. 64ºC for 30 second 4. 72ºC for 40 second 5. repeat 2-4 for 32 cycles 6. 72ºC for 2 minutes
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Procedure
DNA purification from a 1/8” dried blood spot (45 minutes)↓
Tetra-primer ARMS-PCR set-up (30 minutes)↓
Tetra-primer ARMS-PCR amplification (90 minutes)↓
Agarose gel electrophoresis (60 minutes)↓
Gel photography under Blue Light and data analysis (15 minutes)
Notes: 1. The assays for different mutations are performed simultaneously using the same conditions. 2. It costs about $3.00 per genotyping in terms of consumables and reagents.
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GALT mutations
Greg Kopish
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MSUD Y438N Mutations
Greg Kopish
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TRECs are reduced in nearly all forms of SCID
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T Cell Receptor Recombination During Development in the
Thymus
Generation of T cell receptor excision circles (TRECs) occur in >70% of all new (naïve) T cells and can be detected by
PCR
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TaqMan Probe Real-time qPCR
REAL-TIME Quantitative PCR (RT-qPCR)
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TaqMan Probe Real-time qPCR
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TREC measurement using RT-qPCR
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Multiplexing the 384-well Plate
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NBS Card(NSC)
a.k.a. Guthrie Card
Dried blood spots (DBS)
3 mm punch 96 well plate
ExtractDNA
Amplify TREC by real-time
QPCR
Analyze
Scientific Methodology
∆R
n
(am
plifi
cati
on
)
Amplification Plot
TREC plasmid
Standards
ABI 7900HT Fast Real-Time PCR System
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Questions?
Mei Baker
Thank you!