39Difference in size is usually referred to as restriction fragment length polymorphism
(RFLP). The size of the products is easily evaluated by agarose gel electrophoresis.
PCR-based RFLP is used for identifi cation of defi ned mutant alleles leading to com-
plete functional defi ciency of the enzyme CYP2D6.
Real-Time PCR for Detection of CNVs
TaqMan® Copy Number Assays (Life Technologies) are designed to detect and
quantify CNVs and help researchers to better understand these processes by enabling
them to determine CNVs from DNA samples, through a real-time PCR reaction. For
example, study of the locations of CNVs in the human genome shows how they can
be used as biomarkers for susceptibility to human diseases. The assays are used to
validate the discovery of CNVs identifi ed through the use of array-based platforms.
Non-PCR Methods
Arrayed Primer Extension
Arrayed primer extension reaction (APEX) is a straightforward and robust enzy-
matic genotyping method in which hundreds to thousands of variations in the
genome are simultaneously analyzed in a single multiplexed reaction. It differs
from allele-specifi c hybridization in that the genotype information in APEX is
obtained by single base extension, performed by a specifi c DNA polymerase,
together with four different dye terminators (Pullat and Metspalu 2008 ). This
approach ensures highly specifi c discrimination without allele-specifi c hybridiza-
tion, because the primer to be extended anneals just adjacent to the DNA base that
needs to be identifi ed. Selection of primers for specifi c sites or their consecutive
placement in tiled format, shifting them by one base, enables SNP analysis, muta-
tion detection, or resequencing of the DNA template. It also permits the analysis of
insertions, deletions, splice variants, gene copy numbers, or CpG islands within the
genome for gene methylation studies, by performing additional bisulfi te reactions.
Advantages for this method over usual hybridization strategies are:
- Reduced mismatching due to intercession of the polymerase.
- Increased resistance to oligonucleotide failure sequences
- Tolerance of a greater range of hybridization conditions
Enzymatic Mutation Detection
Enzymatic Mutation Detection (EMD) is a streamlined and improved version of the
original Enzymatic Cleavage of Mismatch (EMC) method. It is a fully homoge-
neous, rapid procedure with four steps: gene isolation, hybridization, detection and
Molecular Diagnostic Technologies