Genetics · MU Sofia

10. Methods for detection of deletions and duplications: MLPA; molecular karyotyping (aCGH).

Explains how copy number changes are detected by two molecular methods, MLPA and array-based comparative genomic hybridisation (molecular karyotyping). It takes each method's principle and steps in turn, then its applications, advantages and limitations, with a table comparing the two side by side. It closes with the clinical situations where each test is preferred and how they complement one another.

Inside: 6 sections · 4 labelled pictures · 2 tables · 16 flashcards · 4 min

Topic 10 of 65 in the MU Sofia syllabus for Genetics (Medicine, Year 3). The free AceMed note covers it in 6 sections, about 427 words (4 min of reading), with 16 flashcards.

In this note

  1. Detection of Deletions and Duplications: MLPA & aCGH
  2. I. Introduction
  3. II. MLPA (Multiplex Ligation-dependent Probe Amplification)
  4. III. Molecular Karyotyping (aCGH – Array Comparative Genomic Hybridization)
  5. IV. Comparison: MLPA vs aCGH
  6. V. Clinical Relevance

I. Introduction

  • Genomic deletions and duplications → copy number variations (CNVs).
  • CNVs can involve small regions (single exons) or large chromosomal segments.
  • Detecting them is crucial in:
  • Congenital malformations.
  • Neurodevelopmental disorders (intellectual disability, autism).
  • Cancer genetics.
  • Two powerful molecular methods:
  • MLPA (Multiplex Ligation-dependent Probe Amplification).
  • aCGH (array-based Comparative Genomic Hybridization, or molecular karyotyping).

II. MLPA (Multiplex Ligation-dependent Probe Amplification)

Principle

  • Detects copy number changes in up to 40–50 DNA sequences simultaneously.
  • Works by comparing the relative amplification of DNA probes hybridized to target sequences.
  • Relies on ligation of two probe halves → amplification only if both parts hybridize correctly.

Steps

  1. 1. Hybridization: Two half-probes bind adjacent to each other on target DNA sequence.
  2. 2. Ligation: If both probe halves match perfectly, they are ligated into a full probe.
  3. 3. PCR amplification: Ligated probes are amplified with universal primers (same primer pair for all targets).
  4. 4. Fragment separation: PCR products separated by capillary electrophoresis.
  5. 5. Analysis: Relative peak sizes compared to control DNA →
  • Reduced peak = deletion.
  • Increased peak = duplication.

Applications

  • Genetic disease testing:
  • Duchenne/Becker muscular dystrophy (DMD gene exon deletions/duplications).
  • Hereditary cancer predisposition genes (BRCA1/2 exon deletions).
  • Carrier detection in families.
  • Mosaicism detection in some cases.

Advantages

  • High sensitivity for single-exon deletions/duplications.
  • Relatively cheap and fast.

Limitations

  • Only tests pre-selected target regions (not genome-wide).
  • Cannot detect balanced rearrangements (translocations, inversions).

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