Transcript Tag-ItTM Mutation Detection Kit for CFTR 70+6

Tag-ItTM 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.