1. 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 Pediatrics
University of Wisconsin School of Medicine and Public Health
Science Advisor, Newborn Screening Laboratory
Wisconsin State Laboratory of Hygiene

2. 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

3. Information Resources_NCBI

4. NCBI_OMIM Information includes: Clinical Features Diagnosis
Clinical Management
Pathogenesis
Molecular Genetics
Genotype/Phenotype Correlations
Population Genetics

5. NCBI_PubMed

6. NCBI_Gene

7. NCBI_Gene

8. Information Resources_GeneTests
Information includes:
Summary
Diagnosis
Clinical Description
Differential Diagnosis
Management
Genetic Counseling
Molecular Genetics
Resources

9. HGMD_GALT

10. GALT Mutation Map

11. General Guidelines for PCR_1
Preventing contamination
Cross-contamination
Carryover contamination
Important reaction components
DNA polymerase
Templates
Primers
Deoxynucleoside triphosphate (dNTPs)
Magnesium ions

12. General Guidelines for PCR_2
Cycling parameters
Denaturing
Annealing
Elongation
PCR products detection
Agarose gel electrophoresis

13. 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

14. 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

15. Scheme of tetra-primer ARMS-PCR
Ye, S. et al. Nucl. Acids Res :e88; doi: /nar/29.17.e88
Copyright restrictions may apply.

16. 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

17. 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 µM
MgCl mM
Taq DNA polymerase U
Genomic DNA 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

18. 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.

19. GALT mutations
Greg Kopish

20. MSUD Y438N Mutations
Greg Kopish

21. TRECs are reduced in nearly all forms of SCID

22. 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

23. REAL-TIME Quantitative PCR (RT-qPCR)
TaqMan Probe Real-time qPCR
REAL-TIME Quantitative PCR (RT-qPCR)

24. TaqMan Probe Real-time qPCR
The geometric increase in fluorescence corresponds to the exponential increase in product and is used to determine the threshold cycle or Ct in each reaction
Relative [DNA] during the exponential phase of a reaction is determined by plotting fluorescence against cycle number on a logarithmic scale (so an exponentially increasing quantity will give a straight line).

25. TREC measurement using RT-qPCR

26. Multiplexing the 384-well Plate

27. Dried blood spots (DBS) Amplify TREC by real-time QPCR
NBS Card
(NSC)
a.k.a. Guthrie Card
Dried blood spots (DBS)
3 mm punch
96 well plate
Extract
DNA
Amplify TREC by real-time QPCR
Analyze
Scientific Methodology
∆Rn (amplification)
Amplification Plot
TREC plasmid
Standards
ABI 7900HT Fast Real-Time PCR System

28. Mei Baker mwbaker@wisc.edu
Thank you!
Questions?
Mei Baker