4.5 ARRAY-BASED HYBRIDIZATION Flashcards
Which hybridization method is used when determining target size is not required?
Dot/Slot blots
What types of samples are most efficiently analyzed using dot or slot blots?
Less complex samples (cloned plasmids, PCR products, selected mRNA)
What control is necessary to ensure proper interpretation in dot or slot blot expression analysis?
Amplification or normalization control
What serves as the baseline for interpretation in dot or slot blot hybridization assays?
Negative control (DNA without the targeted sequence)
What is a key advantage of slot blots over dot blots for quantification?
More accurate quantification by densitometry scanning
What can be used for depositing the target DNA or RNA onto the membrane in dot/slot blots?
Various devices, including vacuum systems or a pipet
Why are dot blots especially useful for mutational screening?
Useful for multiple qualitative analyses where many targets are compared
What type of technology allows for the simultaneous study of large numbers of targets or samples?
Array technology
What type of arrays are used to study gene amplification or deletion?
Comparative genome hybridization arrays
Which arrays are applied to the analysis of gene expression, such as RNA or protein?
Expression arrays
Name four approaches to array technology
Macroarrays, microarrays, high-density oligonucleotide arrays, microelectronic arrays
What is the primary advantage of macroarrays over Northern and Southern blots?
Larger sample capacity.
What is a limitation of dot and slot blots regarding the number of genes?
Single gene testing.
Array technique where unlabeled probes are immobilized on the membrane, and the test sample is labeled for hybridization.
Reverse dot blot technique.
Type of blot that allows testing and analyzing larger numbers of samples simultaneously.
Macroarrays
Type of signal typically used to detect hybridization in macroarrays.
Radioactive or chemiluminescent signals.
Device used to read the hybridization of labeled sample material in macroarrays.
Phosphorimager
Material that replaced nitrocellulose or nylon membranes for producing arrays in 1987.
Treated glass.
Technology that allowed the evolution of macroarrays into microarrays by depositing very small target spots.
Improved spotting technology.
Maximum number of targets that can be screened simultaneously on a microarray.
Tens of thousands.
Size of the glass substrate used for microarrays compared to a common laboratory item.
Microscope slide.
System used to automate the deposition of spots on microarrays.
Arrayers
Number of spots that automated systems can place on a glass substrate.
More than 80,000 spots.
Year the first automated arrayer was described by Patrick Brown.
1995
Institution where the first automated arrayer was developed.
Stanford University
Technology used by the first automated arrayers to place probe material.
Pen-type contact
Type of printing systems incorporated into modified arrayers for target deposition.
Ink-jet printing systems
Methods of expulsion used in ink-jet printing systems for target material.
Thermal, solenoid, or piezoelectric
What replaces the larger nitrocellulose membrane in microarrays?
Glass microscope slide.
What is the common term used for the glass slide carrying the array of targets?
Chip
What types of targets are usually immobilized on the glass slide of a microarray?
DNA (cDNAs, PCR products, or oligomers).
How are targets typically arranged on a microarray chip?
In triplicate and spaced across the chip.
What are test samples usually generated from in microarray experiments?
cDNA generated from sample RNA
Besides cDNA, what other types of test samples can be used in microarrays?
Genomic DNA, RNA, or protein.
What is the field that studies the entire genome or sets of related genes?
Genomics
What term describes the complete set of transcripts encoded by a genome?
Transcriptome
What term refers to the set of encoded proteins in an organism?
Proteome
Who predicted that the proteome is likely to be 10 times more complex than the genome?
Stanley Fields
What is the study of entire sets of proteins called?
Proteomics
What technologies facilitate the study of proteomics?
Array technology and mass spectrometry
What method is used to deposit targets for array analysis directly on glass or silicon support?
DNA synthesis
What technology allows for the synthesis of short oligomers (10 to 25 bases long) on high-density arrays?
Proprietary photolithography techniques.
What type of arrays are used for mutation and polymorphism analysis, DNA methylation analysis, and sequencing?
High-density oligonucleotide arrays.
What is required for sample preparation in array analysis for reading microarrays?
Fluorescent labeling of the test sample.
What method is frequently used for labeling RNA in array analysis?
Synthesis of cDNA or RNA copies with labeled nucleotides
What are arrays used to assess gene expression classified as?
Expression arrays
What technique is designed to test DNA for deletions and amplifications in comparative genome hybridization?
Array CGH (comparative genome hybridization).
What advantage does array CGH provide over traditional cytogenetic analysis?
Higher resolution and more defined genetic information.
What is necessary for reading microarrays?
A fluorescent reader and analysis software.
In bead array technology, where are the probes immobilized?
beads
What allows hybridization of targets in bead array technology?
complementary base pairing
How can multiple suspensions be tested simultaneously in bead array technology?
Using a 96-well plate.
How are different probes on beads distinguished in bead array technology?
color-coding the beads with a particular shade of red fluorescent dye.
What color dye is used to label the sample in bead array technology?
Green dye
For what types of targets is bead array technology used?
Protein and nucleic acid targets
What clinical applications are available for bead array systems?
Tests for infectious diseases and tissue typing.
