Table 4.

Advantages and limitations of PCR-based methods identified by Genetic Testing Registry (GTR) screening for relevant biomarkers

PCR-based method	Advantages	Limitations	Reference
ARMS (amplification refractory mutation system, allele-specific PCR)	Simple technique, quick, allele specific Suitable to detect point mutations, small deletions and insertions Determination of haplotypes in clinical diagnostics possible Can also be multiplexed Applicable also in resource-constrained circumstances due to low costs	Inefficient priming can occur	Frayling et al. [18], Yang et al. [68], Little [69], Markou et.al [16]
ASPE	Applicable for high-throughput analysis	Further methods for analysis needed such as electrophoresise or matrix-assisted laser desorption/ionization-time of flight (MALDI) mass spectrometry	Breyer et al. [30]
KASP	Identification of SNPs or InDels, cost-effective, can be multiplexed, applicable also in resource-constrained circumstances due to low costs	Multiplexing limited to single bi-allelic SNP per reaction	Suo et al. [70] , Shitaye [71], He et al. [72]
MLPA	Fast, and relatively simple technique, low costs detection of CNVs and mosaic mutations	Sensitive to DNA quality and quantity Ligation failure due to point mutations in probe binding site and therefore erroneously indication of deletions possible No detection of balanced genomic rearrangements	Katsanis [38], Frayling et al. [18]
PCR–RFLP	Simple and rapid technique for SNP genotyping, detection of point mutations and discrimination of homozygous and heterozygous samples	Limited availability of suitable restriction enzymes	Ota et al. [24]
QF-PCR	Accurate and cost-effective method applied for detection of aneuploidy, detection of mosaicism	No detection of structural abnormalities	Mann and Ogilvie [73], Konjhodzic et al. [74]
qPCR	Closed-tube technique, limits danger of contamination High sensitivity and specificity Multiplexing of reactions and fast analysis possible	Limited combinations of fluorescent dyes for multiplexing	Frayling et al. [18]
RT-PCR/q-RT-PCR	Gene expression analysis	Sensitive to RNA quality	Murphy and Bustin [19]

Most frequenty applied techniques in both listings (‘Testing recommended’/‘Testing required’ and ‘Actionable PGx’) are shaded in bold

ARMS amplification refractory mutation system, ASPE allele-specific primer extension, KASP competitive allele-specific PCR, MLPA multiplex ligation-dependent probe amplification, PCR polymerase chain reaction, QF-PCR quantitative fluorescent PCR, qPCR quantitative real-time PCR, RT-PCR reverse transcriptase PCR, RFLP Restriction Fragment Length Polymorphism Analysis