Transcript Application of Microarray- Based Genomic Technology to Mutation

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.