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DNA Microarray:: A Recombinant DNA Method

DNA microarray is a technique that allows analysis of thousands of genes simultaneously. It involves isolating mRNA from cells, converting it to labeled cDNA, and incubating the cDNA on a microarray chip containing DNA probes for each gene. Unbound cDNA is washed off and a scanner detects which genes were expressed by the presence of fluorescent labels at each location on the chip. The data is analyzed to determine expression levels and ratios between conditions represented by different colors. Microarrays can be used to study gene expression in diseases like cancer and identify mutated genes.

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23 views12 pages

DNA Microarray:: A Recombinant DNA Method

DNA microarray is a technique that allows analysis of thousands of genes simultaneously. It involves isolating mRNA from cells, converting it to labeled cDNA, and incubating the cDNA on a microarray chip containing DNA probes for each gene. Unbound cDNA is washed off and a scanner detects which genes were expressed by the presence of fluorescent labels at each location on the chip. The data is analyzed to determine expression levels and ratios between conditions represented by different colors. Microarrays can be used to study gene expression in diseases like cancer and identify mutated genes.

Uploaded by

Kenty Regina
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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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).

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