EliGene® GIST NGS
EliGene® GIST NGS kit is intended for multiplex PCR of target regions of genes c-KIT (exons 9, 11, 13, 17) and PDGFR (exons 10, 12, 14, 18) allowing detection of polymorphisms included in the amplificated regions.
Principle of the method
EliGene® NGS kits enable multiplex PCR amplification of all required target regions of the gene(s) of interest. The recommended amount of DNA for each multiplex PCR reaction is between 20 and 100 ng of DNA derived from formalin-fixed paraffin-embedded (FFPE) material or from blood. Next, the resulting amplicons are barcoded with MID sequences, and mixed per sample. This sample is quantified and sequenced using a NGS instrument Illumina for massively parallel sequencing (MPS) according to the manufacturer’s instructions. The resulting sequence reads are subsequently analyzed by comparison with the reference sequence of the targeted gene(s) to identify variant positions.
EliGene® NGS kits are used for one or more multiplex PCR reactions resulting in amplification of target regions of one sample. For amplification is used a ready-to-use mastermix containing a hot-start DNA polymerase. The resulting amplicons of each multiplex are diluted 100 fold. In the second step, a second round of PCR is performed using EliGene® Adaptor-IL NGS kit, enabling tagging of all the amplicons to incorporate MIDs and p7 and p5 adaptors required for Illumina MiSeq Sequencing. The resulting tagged amplicons from are mixed per individual applying a predefined assay specific mixing scheme. Each amplicon library is subsequently purified from small residual DNA fragments shorter than 150bp and the DNA concentration is determined using a Real-Time PCR with concentration standards. Next, these purified and individually tagged amplicon libraries are pooled equimolar, resulting in an amplicon pool or sequencing sample, which is then further processed using custom sequencing primers from EliGene® Adaptor-IL NGS kit and sequenced on the Illumina MiSeq. During the sequencing run, different sequence reads are generated on the same cluster in the process of bridge PCR. The positions of the individual custom sequencing primers supplied by EliGene® NGS kit as well as the Illumina sequencing reagents are indicated and clarified.
Primary sample collection, handling and storage
Manual: EliGene Urine Isolation Kit (ELISABETH PHARMACON)
Automatic: MAGNETO BodyFluid DNA/RNA Isolation Kit (for equipment ZEPHYRUS Magneto)
Manual: EliGene® FFPE Tissue DNA Isolation Kit (Elisabeth Pharmacon)
|Ref. No.||Package||Min. package||Price ex. VAT||VAT|
|EG-90068-NGS||24 reactions||1 kit||€ 2 500.00||21 %||Buy|
(published 12.12.2018, size 773.0 kB)
(published 25.07.2018, size 528.2 kB)
(published 25.07.2018, size 527.7 kB)
(published 25.07.2018, size 528.0 kB)