xTAG® Cystic Fibrosis Assay Technology

Step 1 - Nucleic Acid Extraction and Purification
Many commercially DNA extraction kits are available which will provide high quality genomic DNA (UV 260/280 ratios: >1.5-1.7) compatible with the xTAG Cystic Fibrosis 39 kit v2 from human blood specimens. A optimal input quantity of 50ng (range of 10ng to 1.5ug) per sample is required to perform the assay. Step time will vary depending the choice of extraction method.

Step 2 - Multiplex PCR Reaction
The nucleic acid is amplified using one polymerase chain reaction (PCR), a molecular biology technique for rapidly creating multiple copies of DNA. In this case, the technique will make multiple copies of multiple DNA target.

Step 3 - Allele-specific primer extension (for CF)
The amplified DNA is mixed with short sequences (TAG primers) of DNA specific to each target. If the target is present, the primer will bind and will be lengthened through a process called Allele specific amplification. During this extension, a reporter label is incorporated.

Step 4 - Bead Hybridization
Color-coded beads are added to identify the tagged primers. Attached to each differently colored bead is an anti-TAG sequence specific to one of the extended TAG primers. Each anti-TAG only binds to the complementary TAG sequence on the primer.

Step 5 - Addition of Reporter Molecule
The reporter solution is the Streptavidin, R-Phycoerythrin conjugate and will be used to detect the target.

Step 6 - Data Acquisition on Luminex Analyser
Samples are then placed in a Luminex xMAP® instrument where beads are read and analyzed by lasers. The lasers identify the color of the bead and the presence or absence of the labeled target. For each sample, these signals are interpreted by the xTAG® Data Analysis Software to determine whether the wild-type and/or mutant alleles for each of the variations have been detected.