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/