Molecular Techniques
Overview
Molecular techniques are laboratory methods used to analyze DNA and RNA at the sequence level. This chapter covers four core technologies (PCR, Sanger sequencing, next-generation sequencing (NGS), and MLPA) that form the backbone of molecular genetic testing in clinical practice. Each method has a distinct mechanism, a distinct set of detectable variants, and a distinct place in the testing algorithm.
The rapid evolution of molecular testing has transformed clinical genetics. Where a single gene might have been analyzed by Sanger sequencing a decade ago, today's clinicians routinely order multi-gene panels or exome/genome sequencing by NGS. However, older techniques remain clinically relevant: Sanger sequencing is still the gold standard for confirming specific variants, and PCR underlies nearly every molecular method. The principles, strengths, and limitations of each technique are what let you navigate the expanding menu of available tests.
Matching a clinical scenario to the most appropriate molecular test is the core decision-making framework in this chapter. A known familial variant is confirmed by Sanger sequencing. A genetically heterogeneous condition (many possible genes) is best evaluated by an NGS panel or exome. Suspected exon-level deletions or duplications that sequencing would miss are detected by MLPA or CMA.
Key Concepts
- PCR as the foundation: most molecular tests begin with PCR amplification of the target region
- Sanger vs. NGS: Sanger sequences one region at a time with high accuracy; NGS sequences millions of fragments simultaneously
- Detection limits: sequencing detects point mutations and small indels; MLPA and CMA detect exon-level or larger deletions/duplications
- Variant types and test selection: matching the suspected variant type to the technique that can detect it
- Clinical exome/genome: understanding the scope, limitations, and secondary findings associated with broad genomic testing
Amplification
PCR is the fundamental amplification technique that underlies nearly all molecular testing. Standard PCR, real-time/quantitative PCR (qPCR), reverse transcription PCR (RT-PCR), and methylation-specific PCR each have distinct uses. Primer design, thermal cycling, and the principal sources of error (contamination, primer mismatch, allele dropout) are prerequisite knowledge for the other techniques in this chapter.
Copy Number Detection
MLPA detects copy number changes at the exon level, filling the gap between sequencing (which misses large deletions/duplications) and CMA (which may not resolve individual exons). It is commonly used when intragenic deletions account for a substantial fraction of pathogenic variants, such as Duchenne muscular dystrophy and hereditary breast/ovarian cancer.
Together, these techniques form the molecular testing toolkit that genetic counselors must understand to order, interpret, and explain results to patients.
Sequencing
Sanger sequencing uses chain-termination chemistry to determine the nucleotide sequence of a specific DNA region. Despite the rise of NGS, Sanger remains the confirmatory test of choice for individual variants: cascade testing of known familial variants, filling in regions of low NGS coverage, and confirming NGS findings before clinical reporting.
Next-generation sequencing (NGS) is massively parallel sequencing across targeted gene panels, clinical exomes, and whole genomes. Library preparation, sequencing chemistry, and bioinformatic analysis all bear on interpretation, as do the challenges unique to broad genomic testing: variants of uncertain significance, incidental findings, and the ACMG recommendations for reporting secondary findings.