Biology Reference
In-Depth Information
proportion of the total RNA in a cell, it may be necessary to purify the
mRNA away from the rest of the RNA population.
The method for doing this uses a post-transcriptional modification of
eukaryotic mRNA that has, at the 3' end, a long string of ''A''
nucleotides forming the poly(A) tail (see Figure 12-3). The addition of the
poly(A) tail occurs after the RNA has been made (thus the term post-
transcriptional), but before it is used for translation. A string of T
nucleotides, also known as an oligo(dT) molecule, would be
complementary to a poly(A) tail, and oligo(dT) molecules attached
to a solid support can therefore be used to capture the mRNA in a total
RNA sample. This is usually done by putting the solid support with
the bound oligo(dT) into a tube or cylinder, called a column, and
allowing the total cellular RNA mixture to pass through the column. The
mRNA will stick to the oligo(dT) and the remaining RNAs will flow
out of the column. The column is washed with buffer and the purified
mRNA is then collected from the column by altering the salt
concentration of the wash buffer. Alternatively, because of the poly(A)
tail, one can go directly to the next step without isolating mRNA.
We convert mRNA to cDNA through a process called reverse
transcription. This process, diagrammed in Figure 12-3, utilizes a
retroviral enzyme properly called an RNA-dependent DNA polymerase,
FIGURE 12-3.
Reverse transcription of a small mRNA. The reverse transcriptase enzyme is responsible for the
extension of the oligo(dT). Note that the new strand is DNA. The mRNA may be removed
enzymatically or through treatment with a strong base.
