How the xTAG® Cystic Fibrosis 39 Kit v2* and the xTAG® Cystic Fibrosis 60*/71 Kit v2** works:
Nucleic Acid Extraction and Purification Many commercial 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 1 - 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 targets within the CFTR gene.
For 48 samples
CF 39
CF 60/71**
Turn Around Time:
81 min
81 min
Hands On Time:
18 min
18 min
Step 2 - Amplicon Treatment Enzymatic treatment of amplified PCR products cleaves unused reagents (primers and dNTPs) left over after PCR.
For 48 samples
CF 39
CF 60/71**
Turn Around Time:
57 min
57 min
Hands On Time:
20 min
20 min
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 extension. During this extension, a reporter label is incorporated.
For 48 samples
CF 39
CF 60/71**
Turn Around Time::
81 min
91 min
Hands On Time:
20 min
30 min
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.
For 48 samples
CF 39
CF 60/71**
Turn Around Time::
38 min
42 min
Hands On Time:
5 min
9 min
Step 5 - Addition of Reporter Molecule The reporter solution is the Streptavidin, R-Phycoerythrin conjugate and will be used to detect the target.
For 48 samples
CF 39
CF 60/71**
Turn Around Time::
17.5 min
21 min
Hands On Time:
2.5 min
6 min
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.
For 48 samples
CF39
CF 60/71**
Turn Around Time:
30 min
55 min
Hands On Time:
5 min
5 min
Turn Around Time And Hands On Time
*IVD cleared assay
**xTAG® Cystic Fibrosis (CFTR) 71 kit v2 is available only outside of the U.S.
**Products listed are region specific and intended for residents of a particular country/region; this site contains information on devices that may not be approved in some countries/regions, therefore please contact Luminex to obtain the appropriate product info for your country of residence.
