Methodologies
CTGT uses complementary methodologies to attain the most sensitive mutation detection results available:
1. DNA Sequencing
Direct DNA sequencing of PCR products generated from genomic DNA is performed for all tests offered by Connective Tissue Gene Tests. In all instances, sequencing of exons and exon-intron boundaries is performed for all genes. This is the gold standard for mutation detection in genes and is highly sensitive for point mutations, splice site mutations and small exonic deletions, insertions and indels. Connective Tissue Gene Tests’ many years of experience with this approach combined with its extensive mutation and polymorphism databases ensure very high test sensitivity.
2. Deletion / Duplication Analysis
While extremely sensitive, direct DNA sequencing does not detect all types of mutations that may be clinically relevant, including some duplications and deletions. Following extensive evaluation of the methodologies available for CNV detection, Connective Tissue Gene Tests is employing two alternative approaches:
i. CTGT High-Density Targeted (HDT) Array is a custom designed Agilent G3 SurePrint microarray used in conjunction with the Agilent microarray platform. The aCGH technology on which the design is based uses 60mer oligonucleotide sequences designed to selectively complement target areas with an extremely high degree of specificity. High resolution and sensitivity are achieved by assigning a number of probes to each exonic region to allow for a minimum of 3 probes within each 300-500 base section of the genomic DNA sequence, and frequently many more. Non-coding intervening sequences are targeted at a minimum with approximately half of the density used for exonic regions. Many intervening sequences are more densely covered. Each probe used in the design has been thoroughly validated by an in silico bioinformatics process and by in vitro testing and has proven to yield accurate and reproducible results. Though individual copy number values derived from single probes may be highly sensitive, a much higher level of sensitivity is attained by the corroborating data of many closely-spaced probes. The minimum CNV size detected by this high-density array is 300-500 nucleotides, which is the technical limit of the assay using these stringent parameters.
ii. CTGT Real-Time PCR Assay is a highly sensitive, quantitative test designed to detect single exon or multi-exon deletions / duplications. The test may also detect smaller deletions / duplications in exons depending upon the specific location of the CNV in question. CTGT currently offers Real-Time PCR Assay testing for FBN1.
Connective Tissue Gene Tests remains actively involved in evaluating alternative methodologies for mutation detection. These methodologies will be considered for introduction as they mature and are proven reliable, accurate and superior or complementary to existing technology.