LABORATORY TECHNIQUES FOR GENETIC TESTING
DNA sequencing
DNA sequencing is a laboratory technique used to determine the exact sequence of base pairs
(A, C, G, and T) in a DNA molecule. It includes any method or technology that is used to
determine the sequence of the base pairs in a strand of DNA. The base pairs of a small
fragment of DNA are sequentially identified from signals emitted as each fragment is resynthesized from a DNA template strand. Using this process the nucleotide sequence for a
segment of DNA, for example a gene of interest, can be determined.
Next Generation Sequencing
In principle, the concept behind Next Generation Sequencing (NGS) technology is similar to
traditional DNA sequencing. The bases of a small fragment of DNA are sequentially identified
from signals emitted as each fragment is re-synthesized from a DNA template strand. NGS
extends this process across millions of reactions in a massively parallel fashion, rather than
being limited to a single or a few DNA fragments. This advance enables rapid sequencing of
large stretches of DNA base pairs spanning entire genomes. NGS allows for rapid analysis of
multiple genes at a considerably lower cost compared to traditional sequencing techniques. This
technology has brought affordable exome sequencing to the clinical setting.
New genetic testing panels for hereditary breast, ovarian and other cancers using next
generation sequencing have been introduced. These panels are currently geared toward
individuals who test negative for high penetrance genes or have a family history suspicious of
more than one hereditary cancer syndrome. Some current limitations include an unknown
percentage of variants of uncertain significance, uncertainty of level of risk associated with most
of the genes on the panels, and a lack of clear guidelines for surveillance measures for carriers
of some mutations.
Rearrangement/Deletion-Duplication analysis
Certain large genomic rearrangements are not detectable by sequence analysis of the coding
and flanking intronic regions of genomic DNA, thereby necessitating supplementary testing in
some cases. A variety of methods that may be used to identify deletions/duplications include
quantitative PCR, long-range PCR, multiplex ligation-dependent probe amplification (MLPA) and
chromosomal microarray (CMA) that includes the gene of interest.
Family-specific mutation analysis
A family-specific disease-causing mutation is a sequence alteration observed that causes or
predisposes to a particular disease. This testing is for the specific disease-causing mutation
previously identified in a family member.