Transcript DNA Microarray:

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/