Important Terms Related to Microarrays

DNA Microarrays

Gene Expression

• With only a few exceptions, every cell of the body contains a full set of chromosomes and identical genes.
• Gene expression is a highly complex and tightly regulated process that allows a cell to respond dynamically both to environmental stimuli and to its own changing needs.
• This mechanism acts as both an “on/off” switch to control which genes are expressed in a cell as well as a “volume control” that increases or decreases the level of expression of particular genes as necessary.

Central Dogma of Molecular Biology

Central Dogma of Molecular Biology — image from http://en.wikipedia.org/wiki/Image:CDMB2.png Central Dogma of Molecular Biology — image from http://en.wikipedia.org/wiki/Image:CDMB2.png

Transcription

Transcription is the process by which the information contained in a section of DNA is transferred to a newly assembled piece of messenger RNA (mRNA). Transciption is consisted of three steps:

Initiation

Transcription Initiation Transcription Initiation

Elongation

Transcription elongation Transcription elongation

Termination

Transcription termination — image from http://en.wikipedia.org/wiki/Image:Simple_transcription_initiation1.svg Transcription termination — image from http://en.wikipedia.org/wiki/Image:Simple_transcription_initiation1.svg

mRNA

Messenger ribonucleic acid (mRNA) is a molecule of RNA encoding a chemical “blueprint” for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes.

Human mRNA structure — http://en.wikipedia.org/wiki/Image:MRNA_structure.png Human mRNA structure — http://en.wikipedia.org/wiki/Image:MRNA_structure.png

Polyadenylation

• Polyadenylation is the covalent linkage of a polyadenylyl moiety to a messenger RNA molecule at 3’ end (3′ poly(A) tail).
• Protecting mRNA from degradation by exonucleases.
• Important for transcription termination, export of the mRNA from the nucleus, and translation.
• Occurs during and immediately after transcription of DNA into RNA.

Polyadenylation — http://en.wikipedia.org/wiki/Image:Polyadenylation.png Polyadenylation — http://en.wikipedia.org/wiki/Image:Polyadenylation.png

Reverse Transcription

• Reverse Transcription is the transfer of information from RNA to DNA (the reverse of normal transcription).
• This is known to occur in the case of retroviruses, such as HIV, and, in higher eukaryotes, in the case of retrotransposons.
• It is not, however, the general case in most living organisms.

Reverse transcription in RNA Virus — image from http://en.wikipedia.org/wiki/Image:RetroTranscription.jpg Reverse transcription in RNA Virus — image from http://en.wikipedia.org/wiki/Image:RetroTranscription.jpg

Complementary DNA (cDNA)

• cDNA is DNA synthesized from a mature mRNA template in a reaction catalyzed by the enzyme reverse transcriptase.
• This enzyme operates on a single strand of mRNA, generating its complementary DNA based on the pairing of RNA base pairs (A, U, G and C) to their DNA complements (T, A, C and G respectively).
• We can use oligo dT(n) or random oligo as primer.

cDNA synthesis by Reverse Transcriptase — image from http://8e.devbio.com/images/ch04/0405fig2.jpg cDNA synthesis by Reverse Transcriptase — image from http://8e.devbio.com/images/ch04/0405fig2.jpg

Oligonucleotide synthesis

DNA double helix illustration — image from http://www.ctaalliance.org/MCBI/images/oligo.jpg DNA double helix illustration — image from http://www.ctaalliance.org/MCBI/images/oligo.jpg

• Oligonucleotide systhesis is the non-biological, chemical synthesis of defined short sequences of nucleic acids Automated synthesizers allow the synthesis of oligonucleotides (typically single stranded) up to 160 to 200 bases
• Commonly used as primers, probes and to generate restriction sites

Nucleic acid hybridization

• Hybridization is the process of combining complementary, single-stranded nucleic acids into a single molecule.
• Nucleotides will bind to their complement under normal conditions, so two perfectly complementary strands will bind to each other readily.
• Southern and Northern blotting are the exist two kind of nucleic acid hybridization

Hybridization of RNA to DNA — image from http://cswww.essex.ac.uk/staff/W.Langdon/genechip/hybridization_of_taggeed_probes.gif Hybridization of RNA to DNA — image from http://cswww.essex.ac.uk/staff/W.Langdon/genechip/hybridization_of_taggeed_probes.gif

Fluorescence

illuminated pcr products on gel electrophoresis — image from http://en.wikipedia.org/wiki/Image:AgarosegelUV.jpg illuminated pcr products on gel electrophoresis — image from http://en.wikipedia.org/wiki/Image:AgarosegelUV.jpg

Fluorescence is a luminescence that is mostly found as an optical phenomenon in cold bodies, in which the molecular absorption of a photon triggers the emission of another photon with a longer wavelength.

Fluorophore

Fluorophore is a component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different (but equally specific) wavelength.

DNA Labeling

fluorescent image — image from http://www.proteome.org.au/images/UserUploadedImages/saturation-dige.jpg fluorescent image — image from http://www.proteome.org.au/images/UserUploadedImages/saturation-dige.jpg

• DNA Labeling is an addition of a chemical into DNA strand to make them visualizable.
• A label can be a radioactive compound or a fluorophore.
• The most famous fluorophore family used in Microarray experiment is Cyanine (Cy),there are two mostly used of Cy compounds, Cy3 and Cy5.

cyanine-3 (Cy3) and cyanine-5 (Cy5) structure cyanine-3 (Cy3) and cyanine-5 (Cy5) structure

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