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Application of Microarray-Based Genomic Technology to Mutation Analysis and
Microbial Detection
Chapter 14Jizhong Zhou and Dorothea K. Thompson
14.1 INTRODUCTION
• DNA or oligonucleotide microarrays.
• Gene expression profiling and genetic
mutation analysis.
• Single nucleotide polymorphisms (SNPs).
• Multiple mutations, insertions, deletions, and
rearrangements
• Adapting microarray hybridization
14.2 OLIGONUCLEOTIDE MICROARRAYS FOR MUTATION ANALYSIS
• SNPs are the most frequent type of variation.
• One nucleotide difference in every 1,000
between any two copies of a chromosome.
• Directly affect protein structure or gene
expression levels.
Cont.,
• Inheritance of SNPs is very stable,
• Minisequencing,
• Molecular beacons,
• Oligonucleotide ligation,
• 5’ exonuclease assays,
• Large-scale sequence comparisons and mutational
analyses.
• Differential hybridization
14.2.1 Microarray-based Hybridization Assay with Allele-specific Oligonucleotides
• Distinguish between homozygous and
heterozygous allelic variants in diploid genomes.
• Differential hybridization with allele-specific
oligonucleotide (ASO),
• Depends on probe characteristics and detection conditions.
Probe and Array Design
• Stability depends on probe characteristics and hybridization conditions.
• Comparable melting temperatures, • Probe length,
• Base composition, • Mismatch position
• Shorter probe sequence is desirable - overall lower duplex stability
• Longer probes stable duplexes offer less discrimination,
• Single-stranded DNA affect the choice of probe length.
• High salt conditions can form internal secondary structures.
• Thermodynamic,
• Hybridization at higher temperatures can melt any internal
secondary structures.
• ASO probes are designed to have a length that generally ranges from
15 to 25 bp.
Detect all possible SNP substitutions
• One probe (perfect match or PM) -
perfectly complementary to a short section
of the target sequence,
• Other three probes (mismatch probes or
MM) are identical to the PM except at the
interrogation position
Standard tiling design
• Two sets of probes - complementary to both
sense and antisense strands of the target
sequence.
• Detecting all the substitutions in a target
sequence with N base pairs, 8N probes are
needed.
Probe tiling design
Gain-of-signal Approach
• Compares the hybridization signals obtained with probes
perfectly matching mutant (test) and wild-type
(reference) sequences.
• Scoring the hybridization signal gaining patterns,
• Sequence variations of the test heterozygous mutant
samples can be identified.
• Heterozygous mutant sample is labeled with a fluorescent
dye, eg., CYANINE 5 (CY5).
Gain- or loss-of-signal approach
Gain-of-signal analysis Loss-of-signal analysis with two colors
Detection of variations through loss-of-signal analysis
Loss-of-signal Approach
• Quantifying the relative losses of the
hybridization signals.
• 50% of the signal intensity lost for a
heterozygous sequence change,
• Complete signal loss will be observed for a
homozygous change.
Technical Challenges
• Reducing false negative and false positive errors,
• Specificity and sensitivity of array-based assays,
• Tetra-methyl-ammonium chloride alleviate the effects of
nucleotide sequence,
• Suboptimal conditions are needed,
• Secondary structures makes the hybridization less
predictable,
14.2.2 Microarray-based Single-base Extension for Genotyping
• Optimal signal intensities and maximum discrimination.
• Minisequencing.• Detection of primer anneals to the target
nucleotide acid sequence. • All SNPs can be discriminated with optimal
discrimination. • Arrays with high probe redundancy are not
required.
Microarray-based Allele-specific Primer Extension
• Two allele specific oligonucleotide probes from both
strands are designed to terminate at the base 5’ to a SNP,
• Validated with genomic fragments containing nine
human disease mutations (Pastinen et al., 1997, 2000).
• 10-fold improvement in discriminating genotypes
• Determine the base composition of the target
nucleotide adjacent to the 3’-end of each probe.
Dideoxyribonucleotide triphosphates, labeled with
different fluorescent dyes
Probes are attached to the array surface via a 5’-linkage
Hybridized with the probes on the microarrays
Hybridized target sequences and oligonucleotide probes
serve as templates
Extension of primers for single-base
Determined with a fluorescence microscope
Microarray-based Tagged Single-base Extension
• Combines microarray hybridization with
single-base extension.
• Unique sequence tags attached to locus-
specific primers.
• Detected by single-base extension using bio-
functional primers
Microarray-based single base extension
14.2.3 Microarray-based Ligation Detection Reaction for Genotyping
• Genotyping sequence variations,
• Single-base mismatch prevents ligation,
• A G/T mismatch at the 3’- end to be ligated
inhibits the reaction by up to 1,000-fold.
14.3 MICROARRAYS FOR MICROBIAL DETECTION IN NATURAL ENVIRONMENTS
• Limitations of Conventional Molecular Methods for MicrobialDetection :
• Majority of naturally occurring species are not culturable,
• Detection and characterization of microorganisms in
natural habitats,
• High-throughput,
• Cost-effective assessment tools.
Microbial detection tools need to be:
• (1) Simple, rapid, and hence real-time and field
applicable;
• (2) Specific and sensitive;
• (3) Quantitative;
• (4) Capable of high throughput;
• (5) Cost-effective.
14.3.2 Advantages and Challenges
• Target and probe sequences can be very diverse
• Analysis of environmental nucleic acids.
• Contamination
• Retrievable biomass is generally low;
• Sensitivity not enough to detect microorganisms
• Not quantitative
14.3.3 Functional Gene Arrays
• Signatures for monitoring the
physiological status and functional
activities.
• Functional gene arrays (FGAs).
• Monitoring gene expression.
Selection of Gene Probes
• 1. Amplify the desired gene fragment from genomic
DNA.
• 2. Recover the desired gene fragments from natural
environments using PCR-based cloning methods.
• 3. Use oligonucleotide probes.
• Sequences that show 0.85% identity can be used as
specific probes for FGAs.
Specificity
• G + C content,
• Degree of sequence divergence,
• Length and secondary structure of the probe,
• Temperature and salt concentrations.
• FGAs consisting of heme- and copper-containing nitrite reductase genes,
ammonia monooxygenase, and methane monooxygenase genes.
• SSU rRNA genes and yeast genes as positive and negative controls.
• Crosshybridization was not observed at either low (45°C) or high (65°C)
stringency.
DNA microarray hybridization
Sensitivity
• Both pure cultures and soil community samples.
•Sensitivity of the 50-mer is 10 times < PCR based FGAs and 100 times < community genome arrays
Genomic DNA from a pure culture of nirS Genomic DNA from surface soil
Quantitation
• Detecting differences in gene expression patterns under
various conditions.
• Signal intensity and target DNA concentration with DNA from
a pure bacterial culture within a range of 1 to 100 ng.
14.3.4 Phylogenetic Oligonucleotide Arrays
• Ribosomal RNA genes.
• Highly conserved and highly variable regions.
• Ideal molecules for microarray-based detection.
• Cells generally have multiple copies of rRNA genes( 0.95%).
• Detection sensitivity will be higher for rRNA genes than for
functional genes.
• Phylogenetic oligonucleotide arrays (POAs).
Challenges of Phylogenetic Oligonucleotide Arrays
• Specificity
• Hybridization.
• Secondary Structure
14.3.5 Community Genome Arrays
• Membrane-based reverse sample genome probing,
• Different from RSGP in terms of the arraying substrate and signal
detection strategies.
• Use nonporous surface for fabrication and fluorescence-based
detection.
• Miniaturized microarray.
• Bacterial artificial chromosomes (BAC)-based cloning approach.
14.3.6 Whole-genome Open Reading Frame Arrays for Revealing Genome Differences and Relatedness
• Closely related based on SSU rRNA gene sequences
• Conservation of gene functions.• Physiological plasticity • Evolutionary processes.• Genome diversity and relation are examined
using the whole-genome ORF array-based hybridization
14.3.7 Other Types of Microarrays
• Cluster analysis of hybridization - higher resolution.
• Random nonamer oligonucleotide microarray - fingerprinting profiles among closely related strains.
• Microarray hybridization-based array - universal nonamer array generate fingerprints from any microorganisms
14.4 SUMMARY
• SNPs the most frequent type of variation in the human
genome and experimental organisms.
• Approaches for design probe and array for hybridization,
• SBE’s need and its types,
• Conventional detection limits,
• Functional and quantitative analysis of result,
• Other types of array.
Thank you
By Prabhakaran