DNA Microarrays and Hybridization Techniques

Short DNA sequences used as probes in microarrays. They can be synthesized on the chip or separately and added later.

A process where DNA probes are built, one nucleotide at a time, on a chip using photolithography.

A process using light to activate specific areas of a chip for adding nucleotides during microarray synthesis.

Protective groups on nucleotides during microarray synthesis, preventing unwanted reactions until activated by light.

A process where cDNA copies of mRNA are marked with fluorescent tags (Red or Green) to distinguish between different mRNA samples.

The process of combining labeled cDNA with the probes on the microarray, where complementary sequences bind.

The analysis step where the microarray is scanned to detect the fluorescent signals from the bound cDNA.

Interpreting the results from the fluorescent signals on the microarray to understand gene expression patterns in different conditions.

The process of separating double-stranded DNA into single strands, usually achieved by heat or high pH.

The temperature at which half of the DNA molecules in a solution are denatured or single-stranded.

Factors like GC content, salt concentration, DNA length, and modifications can influence the melting point of DNA.

Calculating Tm is crucial in various techniques like Northern, Southern blot analysis and PCR, helping ensure optimal conditions.

For probes 14 to 70 base pairs long, a formula using sodium concentration, GC content, and probe length can be used.

Multiple methods exist for calculating Tm, each tailored for different probe lengths or conditions.

Calculating Tm is essential for designing and analyzing microarrays, ensuring accurate and reliable gene expression measurements.

Online tools and software are readily available to aid in accurate Tm calculation, saving time and effort.

The method used to identify and analyze the signal produced by a probe attached to a target molecule, such as DNA or RNA.

A technique used to detect specific RNA molecules in a sample by separating the RNA by size, transferring it to a membrane, and then probing with a labeled molecule that binds to the target RNA sequence.

A high-throughput technology used to study the expression of thousands of genes simultaneously by using DNA probes attached to a solid surface, allowing for the analysis of gene expression patterns in different conditions.

Microarrays that use complementary DNA (cDNA) probes, which are DNA copies of mRNAs, to detect gene expression levels.

Microarrays that use short, synthetic DNA sequences (oligos) as probes to detect gene expression levels.

The process of combining labeled cDNA (or other nucleic acids) with the probes on a microarray, where complementary sequences bind and form stable pairs.

Oligos, short DNA sequences used as probes in microarrays, can be synthesized in two ways: longer ones made separately and added to the chip, or shorter ones synthesized directly on the chip.

Photolithography uses light to activate specific areas of a chip for adding nucleotides during oligo synthesis. This allows for precise placement of oligos on the chip.

Blocking groups are protective chemicals attached to nucleotides during oligo synthesis. They prevent unwanted reactions until activated by light, ensuring precise nucleotide addition.

Microarray experiments involve several steps: 1) loading probes onto the chip, 2) extracting mRNA from different conditions, 3) labeling cDNA with fluorescent tags, 4) hybridizing labeled cDNA, 5) scanning for fluorescence, 6) analyzing data.

After hybridization, the microarray is scanned to detect the fluorescent signals from bound cDNA. These signals are then analyzed to understand gene expression patterns under different conditions.

The data from microarray experiments is analyzed to identify genes with changed expression levels between different conditions. This helps understand biological processes and potential disease mechanisms.

The process of separating double-stranded DNA into single strands, usually done with heat or a high pH.

The temperature at which half of the DNA molecules in a solution are denatured (become single-stranded).

Calculating Tm helps ensure accurate and reliable gene expression measurements by optimizing conditions for hybridization and probe design.

There are various methods, but a common one for probes is: Tm = 81.5 + 16.6(log ([Na+]) +.41*(%GC) – 600/length). [Na+] is the molar sodium concentration, (%GC) is the GC ratio, and length is the probe length.

The process of measuring the amount of specific mRNA molecules present in a sample, reflecting the level of gene activity.

The method used to identify and analyze the signal produced by a probe attached to a target molecule, such as DNA or RNA.

Analyzing the expression levels of a large number of genes simultaneously to get a comprehensive view of cellular activity.

Microarrays are used to compare gene expression levels between different conditions, such as before and after a treatment or in different cell types.