Tag-It TM Mutation Detection Kit for CFTR 70+6 Lorraine Fernandez August 6, 2008

Testing Overview The Tag-It Mutation Detection Kit for CFTR 70+6 is comprised of two assays: Tag-It Cystic Fibrosis and Cystic Fibrosis B. Each sample is analyzed with both assays and analyzed consecutively on the Luminex 100 xMAP system.

The Tag-It Cystic Fibrosis assay screens for the currently recommended 23 mutations and 4 variants, plus 1078delT, I148T, and 15 of the world’s most common and and North American’s prevalent mutations. The Tag-It Cystic Fibrosis B assay screens for and additional 31 mutations and 1 variant (D1270N).

Clinical Significance Cystic Fibrosis is an autosomal recessive disorder affecting 30,000 children and adults in the United States. Cystic Fibrosis occurs when a patient has 2 mutations (could be 2 different mutations) in the gene for CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) protein which enhances the secretion of chloride in mucus producing epithelial cells.

Over 10 million Americans are carriers (1 in 31). In non-Hispanic Caucasians, the carrier frequency is 1 in 25. Molecular testing is the only testing available to detect carriers.

Specimen Requirements Testing requires whole blood collected in EDTA (lavender top tube). 100ul of sample is used for DNA extraction on the Roche Magnapure. A total of 35 ng of gDNA is required for analysis (25 ng for Cystic Fibrosis and 10 ng for Cystic Fibrosis B).

Cystic Fibrosis PCR Make up PCR Mix for both A and B. Master Mix contains DNase free water, 10X PCR Buffer, 50 mM MgCl2, Cystic Fibrosis PCR Mix or Cystic Fibrosis Mix B, and Platinum Taq DNA Polymerase. For Cystic Fibrosis PCR: add 20 ul of Master Mix and 5 ul of sample For Cystic Fibrosis B PCR: add 8 ul of Master Mix and 2 ul of sample Place tubes in thermocycler. PCR takes approximately 1 hour. Amplimer sizes range from 179 bp to 465 bp.

Amplicon Treatment Shrimp Alkaline Phosphatase (SAP) inactivates any remaining nucleotides (especially dCTP). Exonuclease I (EXO) degrades any primer left over from the PCR reaction. For Cystic Fibrosis PCR: Add 3.5 ul of EXOSAP to PCR reaction (25 ul) For Cystic Fibrosis B PCR: Add 1.4 ul of EXOSAP to PCR reaction (10 ul)

Place tubes in thermocycler. Program takes approximately 45 minutes.

Multiplex ASPE Make up Cystic Fibrosis ASPE Master Mix for both A and B. ASPE Master Mix DNase free water, 10X PCR Buffer, 50 mM MgCl2, Cystic Fibrosis PCR Mix or Cystic Fibrosis Mix B, and Platinum Taq DNA Polymerase. For Cystic Fibrosis PCR: add 15 ul of ASPE Mix to 5 ul of treated PCR product. For Cystic Fibrosis B PCR: add 13 ul of ASPE Mix to 5 ul of treated Cystic Fibrosis PCR AND 2 ul of treated Cystic Fibrosis PCR B

Place tubes in Thermocycler: Program takes approximately 82 minutes.

Bead Hybridization Thaw beads (86 bead population used for A and 64 bead population for B). Vortex and sonicate two times. Add 45 ul of beads to 5 ul of ASPE product. Place tubes in Thermocycler: Program takes approximate 62 minutes.

Wash Beads Prewet filter plate with wash buffer on vacumn manifold system. Add 100ul of wash buffer to the hybridized samples. Then transfer entire contents to the filter plate. Apply vacumn to remove liquid. Add 200 ul of wash buffer to wells. Again apply vacumn to remove liquid. Blot to remove residual liquid.

Detection Add 150ul of reporter solution (Steptavidin, R-Phycoerythrin conjugate) to each well. Cover the plate and incubate for 15 minutes. Place filter plate on Luminex plate holder. Calls are made by comparing the signal to the background.