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Lecture

Lecture 23: "DNA Technologies II"

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Department
Biology
Course
Biology 1002B
Professor
Tom Haffie
Semester
Fall

Description
Biology Lecture No. 23: DNA Technologies II rd Monday April 23 , 2012 RECALL: -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 amplified. 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 be exact). -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 additional PCR. 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 gene
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