Essential Cell Biology 5th edition

340 CHAPTER 10 Analyzing the Structure and Function of Genes
Figure 10–9 Complementary DNA (cDNA)
is prepared from mRNA. Total mRNA
is extracted from a selected type of cell,
and double-stranded complementary
DNA (cDNA) is produced using reverse
transcriptase (see Figure 9−30) and DNA
polymerase. For simplicity, the copying
of just one of these mRNAs into cDNA is
illustrated here. Following synthesis of the
first cDNA strand by reverse transcriptase,
treatment with RNAse leaves a few RNA
fragments on the cDNA. The RNA fragment
that is base-paired to the 3ʹ end of the first
DNA strand acts as the primer for DNA
polymerase to synthesize the second,
complementary DNA strand. Any remaining
RNA is degraded during subsequent
cloning steps. As a result, the nucleotide
sequences at the extreme 5ʹ ends of the
original mRNA molecules are often absent
from cDNA libraries.
5′
5′
5′
3′
5′
3′
mRNA
cDNA
residual
RNA primer
cells in culture
LYSE CELLS AND
PURIFY mRNA
HYBRIDIZE WITH
POLY T PRIMER
mRNA
MAKE DNA COPY WITH REVERSE
TRANSCRIPTASE TO FORM RNA/DNA
DOUBLE HELIX
PARTIALLY DEGRADE RNA
WITH RNAse
SYNTHESIZE A COMPLEMENTARY
DNA STRAND USING DNA POLYMERASE
AAAAAAA 3′
AAAAAAA
3′
3′ TTTTTTT
T T T T T T 5′
poly T primer
AAAAAAA
3′
TTTTTTT
T T T T T T 5′
AAAAAAA
3′
TTTTTTTT
T T T T T 5′
5′
3′
AAAAAAA
3′
T T T T T T T 5′
double-stranded complementary DNA (cDNA) molecule
There are several important differences between genomic DNA clones
and cDNA clones. Genomic clones represent a random sample of all of
the DNA sequences found in an organism’s genome and, with very rare
exceptions, will contain the same sequences regardless of the cell type
from which the DNA came. Also, genomic clones from eukaryotes contain
large amounts of noncoding DNA, repetitive DNA sequences, introns,
regulatory DNA, and spacer DNA; sequences that code for proteins will
make up only a few percent of the library (see Figure 9−33). By contrast,
cDNA clones contain predominantly protein-coding sequences, and only
those sequences that have been ECB5 transcribed e10.12/10.09 into mRNA in the cells from
which the cDNA was made.
As different types of cells produce distinct sets of mRNA molecules, each
yields a different cDNA library. Furthermore, patterns of gene expression
change during development, so cells at different stages in their development
will also yield different cDNA libraries. Thus, cDNAs can be used to
assess which genes are expressed in specific cells, at particular times in
development, or under a particular set of conditions.
Hybridization Provides a Sensitive Way to Detect
Specific Nucleotide Sequences
Thus far, we have been talking about large collections of DNA fragments.
For many studies, however, investigators wish to identify or
examine an individual gene or RNA. Fortunately, an intrinsic property of
nucleic acids—their ability to form complementary base pairs—provides
a convenient and powerful technique for detecting a specific nucleotide
sequence.
To see how, let’s look at a molecule of double-stranded DNA. Under normal
conditions, the two strands of a DNA double helix are held together by
hydrogen bonds between the complementary base pairs (see Figure 5−4).