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