Biology Lecture No. 23: DNA Technologies II
Monday April 23 , 2012
-In order to determine the unknown Vaucheria sequence, multiple sequences of related algae were
aligned. This yields identifiable regions of high sequence conservation; sequences that correlate
perfectly with that of Vaucheria.
-Based on two regions of high sequence similarity, degenerate primers can be designed that encompass
all the possible combinations of the identified DNA sequences. The observed region of DNA is then
Reverse Transcription (RT) - PCR:
-Transcript abundance refers to the amount of a certain mRNA observed; in this case the transcript
abundance of psbO. As the Polymerase Chain Reaction (PCR) amplifies double-stranded DNA, a more
specific process known as Reverse Transcription PCR is needed to amplify single-stranded RNA.
-The first step is to isolate the total RNA (found in either Elysia or Vaucheria), which is mostly rRNA as
opposed to mRNA. Next it is necessary to reverse transcribe that RNA in order to obtain complementary
DNA (cDNA), a single-strand piece of DNA out of an RNA template. The enzyme used in this reverse
transcription is reverse transcriptase.
-This cDNA is double-stranded (half-DNA and half RNA) and can now be used in the PCR process in order
to be amplified. As the mRNA strand contains a poly-adenine tail, DNA primers can be designed (poly-
thymine) in order to bind to the 3’ end of mRNA, which reverse transcriptase can synthesize the
remainder of the cDNA. PCR can then yield a 452 base-pair product.
Identify PCR Product:
-In order to identify whether or not the amplified strands are of the desired psbO, one must excise the
PCR band from the gel, sequence it, and then BLAST the sequence.
Northern Blot Analysis:
-A Northern blot analysis is now needed for showing real expression in Elysia by using the 452 base-pair
product of PCR as the probe. This analysis is looking at total mRNA run on a gel. Through hybridization, it
is observed that Elysia does in fact have nearly the same consistency of bands as Vaucheria does (2 two
-This correlation between Vaucheria and Elysia would be easier to notice if there was a negative control
from which to compare; an organism (preferably a mollusc) which does not carry the psbO gene. Obtaining Entire PsbO gene:
-It is important to note that the 452 base-pairs sequence does not represent the entire transcript
because the primers are from the two regions that are highly conserved, which is found in the middle of
the strand. In order to definitively characterize the entire gene, the “ends” (the rest of the sequence)
must be known.
-These ends can be amplified and thus identified by attaching one primer that binds to the 3’ end of the
original mRNA strand and one primer that binds to the 3’ end of the 452 base-pair sequence on the
complementary DNA strand.
-The other (left) end can be amplified by attaching one primer that binds to the 5’ end of the 452 base-
pair sequence on the original mRNA strand and one primer that binds to the 5’ end of the
complementary DNA strand. The amplification of the entire 962 base-pair sequence is achieved by
Further PCR & RT-PCR:
-Once the entire sequence is identified, homologous primers can then be designed; primers where the
sequence is perfect for finding psbO. The template for PCR is genomic DNA. Starting with small amounts
of genomic DNA, primers are used for the photosynthetic gene, psbO. No requirement for reverse
transcriptase is necessary.
-And finally, the presence of the nuclear gene for psbO is discovered in not just Vaucheria, but in egg
and adult stage Elysia as well. Since the psbO gene is found in the eggs of Elysia, it can be said that this