DNA Microarray: A Recombinant DNA Method
Basic Steps to Microarray: • Obtain cells with genes that are needed for analysis. • Isolate the mRNA using extraction buffer. • Remove the buffer with RNA. • The mRNA will be 5’-3’ with repeating AAA..end. • Convert each mRNA into colored cDNA. • Targets labeled with fluorophores.
Basic Steps to Microarray: • With mRNA degraded reverse cDNA created. • Mix colored samples of cDNA.
Microarray or DNA Chip: • Every spot on the chip represents a different coding sequence from different genes. • Each spot on the chip is made of a DNA probe that can pair with the cDNA that was created.
Basic Steps to Microarray: • Incubation with the mixed cDNA and the chip DNA will yield some pairing. • Wash off unbound cDNA to see what has bound to the microarray.
Visualizing Bound cDNA: • The slide with the microarray chip is placed inside a dark box where it is scanned with a high resolution laser that detects the bound fluorescent labels. • The information and images are then sent to the computer for analysis.
Analyzing the Data: • Creates a ratio image. • Green images signal expression in one condition. • Red images signal expression in one condition. • Yellow images signal expression in both conditions.
Capacities: • Not limited to human genetic material. • Can be used for all species. • Can display thousands of different genes. • Pending chip size. • 100’s nucleotides (<1 cm^2) • 100,000’s nucleotides (about 10 cm^2) • Allows the study of multiple genes at once.
Types of Microarray: • Pre-synthesized nucleic acids. • Bought cDNA. • Oligonucleotide synthesis in situ. • Creating cDNA in question.
Microarray Application: • Identifying gene’s and gene mutations of different types of diseases such as cancer. • Identifying the expression level, or quantity of genes (mutated or not).
Definitions: • Target - the nucleic acid (cDNA) sample who’s identity and quantity are being measured. • Fluorophore – usually green and red labels attached to the target to enable visualizing expression. • Microarray works as reverse hybridization method converting from mRNA to cDNA 3’-5’ with TTTT…end. • Probe – an attached nucleic acid with a known sequence (the DNA chip).
Resources: • Strachan, Tom; Read, Andrew. Human Molecular Genetics 3. pp 175-178. New York. Garland Publishing. 2004. • Campbell,A. Malcom. Microarray Animation. Davidson College. 2001. www.bio.davidson.edu/courses/genomics/chip/chip.html • http://cmgm.stanford.edu • http://www.gene-chips.com/