We followed the standard high throughput TILLING protocol for mutation discovery [ 31].
It provides a scientific basis for mutation analysis and subsequently, following the mutation analysis, an intensified follow-up protocol for mutation carriers.
However, the logistics of such an application for this method are unknown, and a recent protocol for mutation screening using this technique indicates that throughput for sample screening (a maximum of 768 samples over three days) [ 44] is unlikely to be capable of matching that of the presented method (approximately 6000 duplicated samples in the same time period).
The OLA was conducted according to the NIH protocol for mutations at HIV-1B protease positions D30N, I50V, V82A, V82S, V82T, I84V, N88D, and L90M as well as reverse transcriptase positions K103N, Y181C, K65R, T215F, T215Y, M184V, and Q151M [ 21, 31].
This assay relied on the same cycling protocol for EG mutation quantification as described previously.
DNA was extracted and amplified at Roche Molecular Systems prior to being sent to a CLIA-certified laboratory (SeqWright, Houston, TX, USA) to be sequenced using a validated protocol for EGFR mutation detection.
The Mx series were the prospectively detected breast cancer cases before MRI was added to the protocol for BRCA1 mutation carriers in 2001, and those subjected to annual Mx alone based on family history before their BRCA1 mutations were detected subsequently.
8 9 In some countries the screening protocol for BRCA1/2 mutation carriers now recommends the avoidance of mammographic screening before age 30 and advises the use of non-ionising radiation imaging techniques (such as magnetic resonance imaging (MRI)) as the main tool for surveillance at young ages.
Protocols for iterative rapid mutation of S. mobaraensis with helium-driven ARTP system and effective tube screening method of the mutants were established.
Following this finding, clinical examination and instrumental surveillance with mammography, magnetic resonance imaging (MRI) and ultrasonography were initiated according to the institutional protocols for BRCA gene mutation carriers.
