Genetic testing techniques, such as polymerase chain reaction (PCR), Sanger gene sequencing, chromosomal microarray, and exome and genome sequencing

The menu of technologies

The menu of diagnostic genomic technologies, and what each one cannot see - the examinable point

Technique	Detects	Blind to
Karyotype	Aneuploidy, large (>5-10 Mb) rearrangements, balanced translocations	Anything smaller; needs dividing cells
FISH	Targeted deletion/duplication, fusion genes (BCR-ABL)	Everything not probed
Chromosomal microarray (CMA)	Copy number variants down to ~50-100 kb, uniparental disomy	Balanced translocations/inversions, point mutations
PCR	Amplifies a target; fragment analysis sizes repeat expansions	Sequence change (unless sequenced)
Sanger sequencing	Point mutations, small indels in one exon/gene; the confirmation standard	Large deletions (reads only the intact allele), repeat expansions, low-level mosaicism
NGS gene panel	Point mutations + indels across many genes; good depth	Genes not on the panel; repeats; some CNVs
Whole exome (WES)	Coding exons only - ~1-2% of the genome	Introns, promoters, repeats, most structural variants, mitochondrial (unless targeted)
Whole genome (WGS)	Coding + non-coding, structural variants, CNV, mitochondrial	Very large repeat expansions (improving); cost, interpretation burden
MLPA	Exon-level deletions/duplications	Point mutations
Methylation studies	Imprinting disorders, Fragile X full mutation	Sequence change
Long-read sequencing	Repeat expansions, complex structural variants, phasing	Cost, availability

*A "normal gene test" is only as good as the technique* - always ask which platform and what it excludes

Diagnostic yield of WES in an undiagnosed suspected monogenic disorder ~25-40%; WGS adds a further ~5-10%
Trio (proband + both parents) testing raises yield and greatly simplifies interpretation (de novo, phase, segregation)
~1-5% of exomes/genomes yield a secondary (incidental) finding on the ACMG actionable gene list

Dideoxynucleotides terminate extension -> fragments of every length -> capillary separation -> read the base order
One amplicon at a time; ~500-1000 bp read; detects mosaicism only above ~15-20%
Still the confirmation method for an NGS variant

Library preparation -> millions of short reads in parallel -> align to a reference -> call variants
Depth of coverage determines sensitivity for mosaicism and CNV calling
Panel -> exome -> genome is a trade-off of breadth vs depth, cost, and interpretive burden
- A larger test finds more VUS, not just more diagnoses

Sizes an amplicon by capillary electrophoresis -> counts repeat number
Triplet-primed PCR or Southern blot needed for very large expansions
*Sanger sequencing is unreliable for large repeat expansions - the polymerase cannot read through* - this is why repeat disorders are PCR/fragment-analysis diagnoses

Clinical question	Test
Recognisable single-gene syndrome	Targeted single gene (Sanger +/- MLPA)
Known familial variant	Targeted variant testing only
Huntington, SCA, myotonic dystrophy, Fragile X	PCR + fragment analysis
Genetically heterogeneous phenotype (cardiomyopathy, epilepsy, neuropathy, aortopathy)	NGS panel
Undiagnosed multisystem disorder, dysmorphism, ID	CMA first, then trio WES/WGS
Recurrent miscarriage, dysmorphic fetus	Karyotype (balanced translocation) + CMA
Suspected mosaicism (TSC, McCune-Albright, overgrowth)	Deep NGS of affected tissue
Haematological malignancy	Karyotype + FISH + targeted NGS panel
Solid tumour treatment selection	Tumour NGS panel, IHC, ctDNA

ACMG 5-tier classification: pathogenic / likely pathogenic / VUS / likely benign / benign
VUS must not drive management and must not be used for predictive testing in relatives
Segregation studies, functional assays and re-analysis over time resolve some VUS
Secondary findings must be discussed before consenting to exome/genome testing

Phenotype first - the request form's clinical description determines the filtering strategy and therefore the result
Trio testing wherever a de novo cause is plausible
Confirm a critical NGS variant by an orthogonal method before acting on it
Re-analysis of a negative exome after 18-24 months yields additional diagnoses as gene-disease knowledge grows
Direct-to-consumer results are not diagnostic - always repeat in a NATA-accredited laboratory before acting (high false-positive rate for rare pathogenic variants)
Refer to clinical genetics for consent, predictive testing, VUS and reproductive counselling

Medicare rebates cover a limited set (some cancer predisposition, tumour biomarker and pharmacogenomic tests); many genomic tests remain hospital- or patient-funded
Genetic test results may not be used in life insurance underwriting for decisions made on or after 8 Oct 2026
Reproductive carrier screening for CF, SMA and Fragile X is Medicare-rebated

Pharmacogenomics: TPMT/NUDT15 (thiopurines), DPYD (fluoropyrimidines), HLA-B57:01 (abacavir), HLA-B15:02 (carbamazepine), CYP2C19 (clopidogrel), CYP2D6
Non-invasive prenatal testing (cell-free fetal DNA) - a screening test, requires diagnostic confirmation
Circulating tumour DNA / liquid biopsy in oncology
Newborn screening (tandem mass spectrometry + targeted genotyping)
Clonal haematopoiesis detected incidentally on tumour or cfDNA panels

Variant classification is dynamic - VUS are reclassified in both directions; establish who re-contacts the patient
Cost per genome continues to fall; interpretation, not sequencing, is now the bottleneck
Reference databases are skewed toward European ancestry - VUS rates are higher in under-represented populations, including Aboriginal and Torres Strait Islander people; interpret with caution and involve clinical genetics
Movement of WGS toward first-line testing in undiagnosed disease and rapid WGS in critically ill neonates (results in days)

10 more sections, plus exam facts

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