Methods of Chromosomal Analysis

Topic Name Introduction Classical Cytogenetic Methods Molecular Cytogenetic Methods Advanced Genomic Methods

Introduction to Methods of Chromosomal Analysis

Chromosomal analysis is a fundamental aspect of genetic diagnosis, providing crucial information about the structure and number of chromosomes in an individual's cells. These techniques have evolved significantly over the years, from basic microscopic observations to highly sophisticated molecular and genomic approaches.

The field of chromosomal analysis plays a vital role in various areas of medicine, including:

• Prenatal diagnosis
• Pediatric genetic disorders
• Reproductive medicine
• Cancer diagnostics and prognostics
• Research into chromosomal structure and function

Understanding the range of available methods, their principles, applications, and limitations is essential for healthcare professionals involved in genetic diagnosis and counseling.

Classical Cytogenetic Methods

Classical cytogenetic methods form the foundation of chromosomal analysis. These techniques primarily involve direct visualization of chromosomes under a microscope.

1. Karyotyping

Karyotyping is the most basic and widely used method of chromosomal analysis.

• Procedure:
  1. Cell culture (usually lymphocytes or fibroblasts)
  2. Arrest cells in metaphase using colchicine
  3. Hypotonic treatment to spread chromosomes
  4. Fixation and staining (typically Giemsa staining)
  5. Microscopic analysis and arrangement of chromosomes
• Applications:
  • Detecting numerical abnormalities (e.g., trisomies, monosomies)
  • Identifying large structural abnormalities (>5-10 Mb)
  • Prenatal diagnosis
• Limitations:
  • Limited resolution (cannot detect small changes)
  • Time-consuming (requires cell culture)
  • Labor-intensive analysis

2. Chromosome Banding Techniques

Various banding techniques have been developed to improve the resolution and specificity of karyotyping.

• G-banding (Giemsa banding): Most common, produces a series of light and dark bands
• Q-banding: Uses quinacrine mustard, similar pattern to G-banding
• R-banding: Reverse pattern of G-banding
• C-banding: Highlights constitutive heterochromatin
• NOR-banding: Identifies nucleolar organizing regions

These techniques allow for more precise identification of chromosomal regions and specific abnormalities.

Molecular Cytogenetic Methods

Molecular cytogenetic techniques combine the principles of molecular biology with traditional cytogenetics, offering higher resolution and specificity.

1. Fluorescence In Situ Hybridization (FISH)

FISH is a powerful technique that uses fluorescent probes to detect specific DNA sequences on chromosomes.

• Procedure:
  1. Prepare chromosome spreads or interphase nuclei
  2. Denature DNA to single strands
  3. Hybridize with fluorescently labeled probes
  4. Wash and counterstain
  5. Analyze under a fluorescence microscope
• Types of FISH probes:
  • Locus-specific probes
  • Centromeric probes
  • Whole chromosome painting probes
  • Telomeric probes
• Applications:
  • Detecting specific microdeletions or microduplications
  • Identifying chromosomal translocations
  • Aneuploidy screening
  • Cancer cytogenetics
• Advantages:
  • Can be performed on non-dividing cells (interphase FISH)
  • Higher resolution than traditional karyotyping
  • Can detect specific known abnormalities

2. Comparative Genomic Hybridization (CGH)

CGH compares the genomic content of two DNA samples, typically a test sample and a reference sample.

• Procedure:
  1. Label test and reference DNA with different fluorochromes
  2. Co-hybridize to normal metaphase spreads
  3. Analyze fluorescence ratios along chromosomes
• Applications:
  • Detecting copy number variations
  • Cancer genome analysis
• Limitations:
  • Cannot detect balanced rearrangements
  • Limited resolution compared to array-based methods

Advanced Genomic Methods

Advanced genomic methods offer high-resolution, genome-wide analysis of chromosomal abnormalities.

1. Chromosomal Microarray Analysis (CMA)

CMA, also known as array comparative genomic hybridization (aCGH), is a high-resolution method for detecting copy number variations.

• Procedure:
  1. Extract and label patient DNA
  2. Hybridize to a microarray containing thousands of DNA probes
  3. Scan and analyze fluorescence intensities
• Types:
  • BAC arrays
  • Oligonucleotide arrays
  • SNP arrays
• Applications:
  • Detecting submicroscopic deletions and duplications
  • First-tier test for developmental delays and congenital anomalies
  • Cancer genome analysis
• Advantages:
  • High resolution (can detect changes as small as 10-20 kb)
  • Genome-wide coverage
  • Does not require dividing cells
• Limitations:
  • Cannot detect balanced rearrangements
  • May detect variants of uncertain significance

2. Next-Generation Sequencing (NGS) Based Methods

NGS technologies have revolutionized chromosomal analysis by providing base-pair level resolution.

• Whole Genome Sequencing (WGS):
  • Sequences the entire genome
  • Can detect single nucleotide variants, indels, and structural variations
  • Highest resolution but computationally intensive
• Whole Exome Sequencing (WES):
  • Sequences all protein-coding regions
  • More focused than WGS, but may miss non-coding variants
• Applications:
  • Detecting complex structural variations
  • Identifying breakpoints in balanced rearrangements
  • Comprehensive genetic analysis in undiagnosed disorders

3. Optical Genome Mapping

A newer technology that provides high-resolution genome-wide analysis of structural variations.

• Procedure:
  1. Label high-molecular-weight DNA at specific sequence motifs
  2. Linearize and image individual DNA molecules
  3. Analyze label patterns to detect structural variations
• Advantages:
  • Can detect balanced and unbalanced structural variations
  • Long-range information (up to megabases)
  • Complements sequencing-based approaches

Methods of Chromosomal Analysis

1. What is karyotyping?
   A technique used to visualize and analyze the complete set of chromosomes in a cell
2. What staining technique is commonly used in traditional karyotyping?
   G-banding (Giemsa banding)
3. What is the resolution of a standard G-banded karyotype?
   Approximately 400-550 bands per haploid set
4. What is Fluorescence In Situ Hybridization (FISH)?
   A cytogenetic technique that uses fluorescent probes to detect specific DNA sequences on chromosomes
5. What is the advantage of FISH over traditional karyotyping?
   It can detect smaller chromosomal abnormalities and specific gene rearrangements
6. What is Comparative Genomic Hybridization (CGH)?
   A molecular cytogenetic method for analyzing copy number variations in the DNA of a test sample compared to a reference sample
7. What is array Comparative Genomic Hybridization (aCGH)?
   A high-resolution, genome-wide screening technique that detects copy number variations using microarrays
8. What is Multiplex Ligation-dependent Probe Amplification (MLPA)?
   A technique used to detect abnormal copy numbers of multiple genomic DNA or RNA sequences
9. What is Quantitative Fluorescence PCR (QF-PCR)?
   A rapid prenatal test for detecting common chromosomal aneuploidies using fluorescently labeled PCR primers
10. What is Spectral Karyotyping (SKY)?
    A molecular cytogenetic technique that uses fluorescently labeled chromosome-specific painting probes to visualize all chromosomes in different colors
11. What is Multicolor FISH (M-FISH)?
    A FISH technique similar to SKY that uses a combination of fluorochromes to uniquely label each chromosome
12. What is Chromosomal Microarray Analysis (CMA)?
    A technique that uses microarrays to detect copy number variations and loss of heterozygosity across the genome
13. What is Next-Generation Sequencing (NGS) in the context of chromosomal analysis?
    High-throughput DNA sequencing technologies that can detect a wide range of genomic variations, including chromosomal abnormalities
14. What is Whole Genome Sequencing (WGS)?
    A comprehensive method of analyzing the entire genomic sequence of an organism
15. What is Whole Exome Sequencing (WES)?
    A technique that sequences all the protein-coding regions (exons) of genes in a genome
16. What is Chromosome Conformation Capture (3C)?
    A technique used to analyze the spatial organization of chromosomes in the nucleus
17. What is Hi-C?
    A high-throughput version of 3C that provides a genome-wide map of chromosomal interactions
18. What is Single-Cell Sequencing?
    A method to analyze the genome or transcriptome of individual cells, useful for studying chromosomal mosaicism
19. What is Optical Mapping?
    A technique that creates high-resolution restriction maps of entire genomes, useful for detecting large structural variations
20. What is Long-Read Sequencing?
    Sequencing technologies that can read long stretches of DNA, useful for detecting complex structural variations
21. What is Chromatin Immunoprecipitation Sequencing (ChIP-seq)?
    A method used to analyze protein interactions with DNA, useful for studying chromosomal proteins and epigenetic modifications
22. What is RNA Sequencing (RNA-seq)?
    A technique used to analyze the transcriptome, which can provide information about gene expression and chromosomal rearrangements
23. What is Methylation-Specific PCR (MSP)?
    A method used to detect DNA methylation patterns, which can provide information about chromosomal imprinting
24. What is Genome-Wide Association Study (GWAS)?
    A method that scans markers across the genomes of many people to find genetic variations associated with a particular disease
25. What is Droplet Digital PCR (ddPCR)?
    A method that provides absolute quantification of target DNA or RNA molecules, useful for detecting copy number variations
26. What is Fiber-FISH?
    A high-resolution FISH technique performed on extended DNA fibers, useful for mapping closely spaced genes or detecting small rearrangements
27. What is Chromosome Flow Sorting?
    A technique used to separate and collect specific chromosomes based on their size and base-pair composition
28. What is Primed In Situ Labelling (PRINS)?
    A technique that combines in situ hybridization with primer extension to detect specific DNA sequences on chromosomes
29. What is Representational Oligonucleotide Microarray Analysis (ROMA)?
    A method that uses oligonucleotide microarrays to detect copy number variations in the genome
30. What is Nanopore Sequencing?
    A third-generation sequencing technology that can sequence long DNA molecules by passing them through tiny pores, useful for detecting structural variations

Further Reading

• Chromosomal Microarray Analysis (CMA) for Developmental Delay, Autism Spectrum Disorders, Intellectual Disability
• Structural variation in the human genome and its role in disease
• Next-generation Cytogenetics: Comprehensive Assessment of Chromosomal Aberrations by Optical Genome Mapping
• Chromosome Analysis - Medical Genetics Summaries