This Application Note describes how to use the Agilent 2100 bioanalyzer to detect mutations in the K-ras and P16 genes.
This document describes a method for identifying point mutations in the K-ras gene codon 12 region and the CDKN2A/P16 gene. The technique utilizes nested PCR followed by restriction enzyme digestion (BstNI) to differentiate between wild-type and mutated DNA sequences based on fragment size.
The analysis is performed using the Agilent 2100 bioanalyzer with the DNA 1000 LabChip kit. This platform allows for the separation of PCR fragments ranging from 135 bp to 106 bp, enabling accurate detection of heterozygous mutations within a 10-15% base pair length resolution. The presence of extra bands on the chip image correlates with mutated DNA sequences, which are subsequently verified by DNA sequencing.
| Resolution for heterozygous mutations | 10-15 % of base pair length |
|---|---|
| Fragment separation range | 135 bp to 106 bp |
| Sample throughput time | 30-40 minute time frame |
Wild-type samples are trimmed by the BstNI enzyme to a 106 bp size, while mutated samples remain at 135 bp.
The Agilent 2100 bioanalyzer with the DNA 1000 LabChip kit is used.