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Chapter 14 Jizhong Zhou and Dorothea K. Thompson

Application of Microarray-Based Genomic Technology to Mutation Analysis and Microbial Detection. Chapter 14 Jizhong Zhou and Dorothea K. Thompson. 14.1 INTRODUCTION. DNA or oligonucleotide microarrays. Gene expression profiling and genetic mutation analysis.

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Chapter 14 Jizhong Zhou and Dorothea K. Thompson

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  1. Application of Microarray-Based Genomic Technology to Mutation Analysis andMicrobial Detection Chapter 14 Jizhong Zhou and Dorothea K. Thompson

  2. 14.1 INTRODUCTION • DNA or oligonucleotidemicroarrays. • Gene expression profiling and genetic mutation analysis. • Single nucleotide polymorphisms (SNPs). • Multiple mutations, insertions, deletions, and rearrangements • Adapting microarray hybridization

  3. 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.

  4. Cont., • Inheritance of SNPs is very stable, • Minisequencing, • Molecular beacons, • Oligonucleotideligation, • 5’ exonuclease assays, • Large-scale sequence comparisons and mutational analyses. • Differential hybridization

  5. 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.

  6. 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

  7. 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.

  8. 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

  9. 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.

  10. Probe tiling design

  11. 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).

  12. Gain- or loss-of-signal approach Gain-of-signal analysis Loss-of-signal analysis with two colors

  13. Detection of variations through loss-of-signal analysis

  14. 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 homozygouschange.

  15. 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,

  16. 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.

  17. Microarray-based Allele-specific Primer Extension • Two allele specific oligonucleotideprobes 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.

  18. 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 oligonucleotideprobes serve as templates Extension of primersfor single-base Determined with a fluorescence microscope

  19. 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

  20. Microarray-based single base extension

  21. 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.

  22. 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.

  23. 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.

  24. 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

  25. 14.3.3 Functional Gene Arrays • Signatures for monitoring the physiological status and functional activities. • Functional gene arrays (FGAs). • Monitoring gene expression.

  26. 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.

  27. 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. • Crosshybridizationwas not observed at either low (45°C) or high (65°C) stringency.

  28. DNA microarray hybridization

  29. Sensitivity • Both pure cultures and soil community samples. Genomic DNA from a pure culture of nirS Genomic DNA from surface soil • Sensitivity of the 50-mer is 10 times < PCR based FGAs and 100 times < community genome arrays

  30. 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.

  31. 14.3.4 PhylogeneticOligonucleotide 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. • Phylogeneticoligonucleotide arrays (POAs).

  32. Challenges of PhylogeneticOligonucleotide Arrays • Specificity • Hybridization. • Secondary Structure

  33. 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.

  34. 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

  35. 14.3.7 Other Types of Microarrays • Cluster analysis of hybridization - higher resolution. • Random nonameroligonucleotide microarray - fingerprinting profiles among closely related strains. • Microarray hybridization-based array - universal nonamer array generate fingerprints from any microorganisms

  36. 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.

  37. Thank you By Prabhakaran

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