Chapter 14 Jizhong Zhou and Dorothea K. Thompson are the most frequent type of variation. • One nucleotide difference in every . • Directly . Cont., • Distinguish between homozygous and heterozygous allelic variants in diploid genomes. • Differential hybridization with allele-specific oligonucleotide (ASO), • Depends on probe characteristics and detection conditions. • 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. • 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 • 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. • Compares the hybridization signals obtained with probes perfectly matching mutant (test) and wildtype (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., Gain-of-signal analysis Loss-of-signal analysis with two colors • 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. • Optimal signal intensities and maximum discrimination. anneals to the target nucleotide acid sequence. • All SNPs can be discriminated with • Arrays with not required. are • 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 • Combines with single-base extension. attached to locusspecific primers. • Detected by single-base extension using • 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. • Majority of naturally occurring species are not culturable, • Detection and characterization of microorganisms in natural habitats, • High-throughput, • Cost-effective assessment tools. • (1) Simple, rapid, and hence real-time and field applicable; • (2) Specific and sensitive; • (3) Quantitative; • (4) Capable of high throughput; • (5) Cost-effective. • Signatures for monitoring • 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. • 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. • 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 • Detecting differences in under various conditions. with DNA from a pure bacterial culture within a range of 1 to 100 ng. • Ribosomal RNA genes. • Ideal molecules for microarray-based detection. • Cells generally have multiple copies of rRNA genes for rRNA genes than for functional genes. • 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 fluorescencebased detection. • Miniaturized microarray. • Bacterial artificial chromosomes (BAC)-based cloning approach. • 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 - . - • 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.