1. PCR and qPCR: a focused question
PCR amplifies a selected DNA region. It can test a known variant, a small target set and some repeat or infectious sequences; qPCR also measures signal over time and can estimate quantity. Its strength is speed and sensitivity, while its central limitation is that it cannot interrogate targets it was not designed to amplify.
2. Sanger: a short sequence at high accuracy
Sanger sequencing reads a limited region and is useful for one gene, a familial variant or focused confirmation. It scales poorly to hundreds of genes, may miss low-level mosaicism and does not replace methods for large rearrangements or complex repeats.
3. SNP chips and chromosomal microarray
A genotyping SNP chip queries preselected common sites and is widely used in DTC products and research. Clinical CMA looks for genomic gains and losses and sometimes regions of homozygosity. Neither equals sequencing: an unknown rare variant between probes can remain invisible.
4. NGS panels: deep, within selected boundaries
A panel sequences tens or hundreds of genes at once, often at high depth, which is useful in inherited disease and oncology. Yet gene lists, exon and intron boundaries, and CNV/fusion algorithms differ. A panel name does not reveal its coverage; the technical specification does.
5. Exome and genome: broader, not omniscient
WES mainly analyses protein-coding regions, while WGS covers coding and non-coding sequence. Genome sequencing is often more uniform and improves some structural-variant detection, but both have difficult regions, depend on coverage and bioinformatics, and may miss expansions, methylation or mosaicism without specialised analysis.
6. RNA, FISH, MLPA and karyotyping answer different questions
RNA sequencing measures expression and can detect fusions or splicing effects. FISH visualises selected rearrangements and defined amplification/copy-number patterns, MLPA measures targeted deletions/duplications, and karyotyping detects large chromosomal changes. These methods are not obsolete; they complement DNA NGS where its physics and design are limited.
7. Methylation, long repeats and long reads
Imprinting disorders and some tumour classifiers require methylation testing. Repeat-expansion disorders need repeat-primed PCR, Southern blot or specialised analysis. Long-read sequencing can resolve repeats, phase and structural variants, but clinical availability and validation remain indication-specific.
8. The whole laboratory chain determines the result
Specimen, transport, extraction, depth, minimum allele fraction, controls, reference build, classification and confirmation all matter. Tests should be compared by intended clinical use and variant class, not by the largest advertised gene count.